vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004
GENETIC VARIATION AND GEOGRAPHICAL DISTRIBUTION OF THE SUBTERRANEAN TERMITE GENUS RETICULITERMESItN Tpx.q,S
JamesW. Austin2,Allen L. Szalanski2,Roger E. Gold3,and Bart T. Fost#
ABSTRACT
A molecular geneticsstudy involving DNA sequencingof a portion of the mitochondrialDNA 165 genewas undertakento determinethe extent of geneticvariation with Reticulitermesspp. and the distribution of Reticulitermesspp. subterraneantermites in Texas.From 42 Texascounties a total of 68 R. flavipes, sevenR. hageni,eight R. virginicus,and nine R. tibialis were identified. No geneticvariation was observedin R. virginicus andR. hageni,while sevenhaplotypes were observedin R. tibialis and 13 for R. flavipes.Among the 13.R.flavipes haplotypes,9nucleotides were variableand genetic variationranged from 0.2 to l.60/o.Phylogenetic analysis did not revealany relationships amongthe R. tibialis arld R. flavipes haplotypes,and there wasino apparentgeographical structureto the haplotypes.The high amount of genetic variation, but a lack of genetic structure in R. flavipes supports the hypothesis that this termite species has been distributedrandomly by mandue to its associationwith structures.
INTRODUCTION
The most abundant native termite in Texas is the subterranean genus ReticulitermesHolgren (Rhiniotermitidae).Four species, the eastern subtenanean Reliculitermesflavipes (Kollar), light southemR. hageniBanks, arid n. nDialis Banks,and dark southernR. virginicus (Banks),are known to occur in Texas(Howell et al. 1987). These speciesare among the most destructiveand costly termites for homeownersand businessesalike, and are of considerableeconomic importance. Su (1993)estimated that over $ I .5 billion is spentannually for termite control in the U.S., of which 80olois spentto control subterraneantermites, More recent estimatesby the National Pest Management Associationsuggest the cost to exceed$2.5 billion annually(Anonymous 2003). While tlrereare no currentestimates of the total economicimpact of Reticulitermesin Texas, Howell et al. (1987) estimated that the costs for termite inspections, treatment of infestations,and repair of damagein Corpus Christi, Texas, alone was $3.7 million annuallyand $30 million annuallyfor the greaterHouston, Texas, area. ln 1979, an attempt to determinethe geographicaldistribution of termites in the stateof Texaswas startedby Howell et al. (1987), and the collection effort has continued to the present.This endeavorprincipally utilizes specimens provided from theprofessional
IIsoptera: Rhinotermitidae 'Department of Entomology,University of Arkansas,Fayetteville, AR7270l 'Department of Entomology,Texas A&M University,College Station, TX77843 pest managementindustry in Texas and specimenswhich are available in the insect museumat TexasA&M University. Correct identification is critical for pest insects,such as termites, which may require very different contol methods dependingon the target species.Identifuing workers is nearly impossible and separatingsoldiers is especially difficult given that precise measurementsare required and overlap may occur between species(Sheffrahn and Su 1994).Difficulties arisein speciesdetermination at individual collectionsites since the majority of the termitesencountered are workers.Finding an alate'in a collection is seasonaland quite rare. Soldiers representonly l-3%i of Reticulitermescolonies and are morphologically variable; use of this castealone can result in equivocal speciesdeterminations. Subtle clinal variations imposed by geographic boundariescan be misleadingin correct speciesdetermination. Molecular geneticmethods are able to differentiate speciesregardless ofthe casteencountered (Szalanski et al. 2003). Also, genetic information obtained from collections is an integral componentto phylogeneticstudies as a whole. Remarkableas it may seem,tlere are currently no known studiesthat have attemptedto look at the extent ofgenetic variation and zubsequentgene flow in Reticulitermesfrom Texas. Information on how geneticvariation is partitionedwithin populationsand among termite speciescan be usefiil for determiningthe extent ofgene flow and for developing molecular diagnostics for identifring species. Previous studies have focused on Reticulitermesspp. from the southeastemUnited Statesand Western Europe (Jenkinset al. 1998,2001; Marini andMantovani 2002). More recently,Austin et al. (2002)included locationswithin Texas and other areas,but additionalpopulations are neededto establish their respectivegenetic parameters. Both cytochromeoxidase II (COII) and 163 rRNA of the mitochondrialDNA (mtDNA) have proved usefrrl for determining phylogenetic relationships of termites (Austin et aI. 2002; Jenkins et al. 1999, 2001; Kambhampatiand Smith 1995, Kambhampatiet al. 1996; Lo et al. 2000; Miura et al. 1998).Using a new molecular diagnostic method for discriminating between closely related Reticulitermesspp. (Szalanskiet al. 2003),we hopeto not only confirm existingdistributions but to expand their known occurrences. For example,within the insect collection at Texas A&M University, College Station, Texas (entowww.tamu.edu/nedresearch/ systematics/collection.html),there are presently 227 Reticuliterzes samples,of which only 96 havebeen classified to species(85% R.flovipes,To/o R. virginicus,TYoR. hageni and 1%oR. tibialis). Fifty-eight percent,representing l3l vials, have not yet been identifiedto species.Identification ofexisting specimens,using moleculartechniques as outlined in this study, from existing collectionssuch as this, can add significant informationon their distribution and gene flow. Additional, information provided by observingthe geneticvariation and gene flow canelucidate existing pattems of migration, potentialhybridization events and general speciation of Reticulitermesspp. in Texas. We investigated the extent of genetic variation within and among Texas Reticulitermestermites, evaluated the utility of these genetic markers for identiffing species,and updatedthe geographicaldishibution oftlese ta: MATERIALS AND METHODS Termiteswere collectedfrom variouslocations in Texasand preservedin 100% ethanol(Table l). In additionto our own collectingefforts, we solicitedthe assistanceof Pest Management Professionals (PMPs) tluoughout the state for the purpose of interpreting the predominant species recovered from infested struchres. PMPs were provided with collection kits and 6ll sampleswere collected throughout the state. A subsample,representative ofvarious geographiczones throughout the state,was usedfor molecularanalysis. Reticulitermes were morphologically identified to specieswhen eitler alates or soldiers were available using the keys of Krishna and Weesner(1969), Scheftahn and Su (1994), Hostettleret al. (1995) and Donovanet al. (2000). For the remainingsamples, species identification was conductedusing DNA sequences(Szalanski et al. 2003). Two additional taxa (Table l) were included as outgrouptaxa to corroborate relationships within the genus for our phylogenetic analysis. Voucher specimens, preservedin 100% ethanol, are maintained at the Arthropod Museum, Departmentof Entomology,University of Arkansas,Fayetteville, AR. TABLE l. Collectiondata, and haplotypes for Texas.Reficuli/ermes and outgroup taxa. Species City CounU Haplotype N R.flavipes CorpusChristi Nueces A 1 Del Rio Val Verde B Carrollton Dallas C Houston Harris c Taylor Williamson c SanAntonio Bexar D Waco McLennan D LakeJackson Brazoria E Dallas Dallas E Friendswood Galveston E Granbury Hood E Beaumont Jefferson E Buffalo Leon E The Woodlands Montgomery E Austin Travis E Hempstead Waller E Lewisville Denton F Odessa Ector F Houston Harris F Houston Harris F Nederland Jefferson F Paris Lamar F Austin Travis F Beeville Bee G Pittsburg Camp G Plano Collin c Dallas Dallas G Rowlett Dallas G Stephenville Erath G Spring Harris G Quinlan Hunt G Beaumont Jefferson G Combine Kaufman G Mabank Kaufman G Jewett Leon G Midland Midland G Livingston Polk G Troup Smith G Arlington Tarrant G Del Rio Val Verde G Mabank Kaufman G Onalaska Polk H Blanco Blanco H Gun Barel City Henderson H The Woodlands Montgomery H Amarillo Potter H Sugarland Fort Bend I Lubbock Lubbock I Baytown Hanis J Magnolia Montgomery J The Woodlands Montgomery J Richardson Dallas J Red Water Bowie K Addison Dallas L Garland Dallas L 2 "| Irving Dallas L Rowlett Dallas L I Fritch Hutchinson L I Kemp Kaufrnan L 2 Midland Midland L I Milano Milam M I Dumas Moore M I R. hageni CollegeStation Brazos HI ) Lewisville Denton HI I Athens Henderson H1 I R. virginicus CollegeStation Brazos V1 Bryan Brazos V1 I Athens Henderson vl 2 R. tibialis Fort Worth Tarrant TI Collin T1 Brackettville Kinney T2 El Paso El Paso T3 New Braunfels Comal T4 Happy Swisher T5 De Soto Dallas T6 Athens Henderson T7 Copt o terme s for mos anus GalvestonIs. Galveston outgroup Heterotermesaureus SantaRita. AZ outsrouD Alcohol-preservedspecimens were allowed to dry on filter paper, and DNA was extractedaccording to Liu and Beckenbach(1992) and Jenkins et al. (1999)on individual whole worker termites with the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis,MN). ExtractedDNA wasresuspended in 50pl of Tris:EDTA andstored at - 20oC.Polymerase chain reaction was conductedusing the primers LR-J-13007(5'- TTACGCTGTTATCCCTAA-3')(Kambhampati and Smith 1995)and LR-N-13398(5'- CGCCTGTTTATCAA,AJMCAT-3')(Simon et al., 1994).These PCR primers ampli$ an approximately428 bp region of the mtDNA 165 rRNA gene. The PCR reactionswere conductedwith lpl of the extractedDNA (Szalanskiet al. 2000), having a profile consistingof 35 cyclesof 94'C for 45s,46oC for 45s and 72oCfor 60s. Amplified DNA from individual termites was purified and concentratedwith minicolumns (Wizard PCRpreps,Promega, Madison, WI) accordingto the manufacturer'sinstructions. Samples were sent to The University of ArkansasDNA SequencingFacility (Fayetteville,AR) for direct sequencing in both directions. DNA sequencesfrom representativesof each haplotypewere submittedto GenBank,accession numbers Ay441975 toAy44l992. DNA sequenceswere aligned using the PILEUPcommand of GCG (Accelrys,San Diego, cA). Mitochondrial DNA haplotypeswere aligned using Macclade v4 (Sinauer Associates,Sunderland, MA). The distancematrix option of PAUP* 4.0b10 (Swofford 2001) was used to calculategenetic distancesaccording to the Kimura 2-parametermodel of sequence evolution (Kimura 1980). Mitochondrial 165 sequencesfrom the Formosantermite, Coptotermesformosanus Shiraki, and Heterotermes aureus (Snyder), GenBank AY380299,were added to theReticulitermes DNA sequencesto act as outgrouptaxa. The DNA sequenceswere aligned by the PILEUP program in GCG (Geneticscomputer Group, Madison, WI) and adjustedmanually. Maximum parsimony analysison the alignmentwas conductedwith PAUP 4.0b10 (Swofford 2001). Gaps were treatedas missingdata. The reliability of treeswas testedwith a bootstraptest (Felsenstein1985). Parsimonybootstrap analysis included 1,000 resamplings and usedthe Branchand Bound algorithmof PAUP. RESULTS DNA sequencingof the l6s rDNA ampliconrevealed that it averaged42g bp in size.The average base frequencies were A = 0.39,C = 0.23,G:0.14, andT = 0.24.From the DNA sequenceanalysis of Reticulitermesfrom 42 Texascounties, a total of 6g R. fl3vipes,7 R. hageni,S R. virginicus,and 9 R. tibiatis were identifiedbased on species diagnosticnucleotide sites from Szalanskietal. (2003)(Table I, Fig. l). No genetic variation was observedin R. virginicus and R. hageni, while seven unique haplotypeswere found in R. tibialis and 13 in R. virgincus (Table l). pairwise Tajima-Neidistances (Tajima and Nei 1984)among Reticulitermes taxarangedfrom 5.7Yo betweenR. flavipes andR. hageni,to 8.3o/obetween R. flavipes ond R. tibialis.A total of nine nucleotidesites were variableamong the 13 R. flavipes haplotypes(Table 2), and geneticvariation among the R. flavipes haplotypesranged from 0.2 to l.6yo. within R. tibialis a total of six nucleotidesites were variableamong the sevenhaplotypes, and variationamong the R. tibialis haplotypesranged from 0.2to l.2yo. TABLE 2. Haplolpe variationat nine nucleotidesites among .R. j/lavjpes from Texas. Haplotype 131 158 168 179 206 236 270 271 274 A B tcc*'trf,tilr.+ c A G'r.T,| C +*+ ,1. D A G * '1. c cr.G E A +*,ti,t *r.G '1. F A GG,I,I C ,. G G G***C *CG H G**,i c {. ,r. G I G*TCc :**G J G***c 'r*G K G{.TCC +:tG L G,iT* c 'r.*G M f,!** c *+G The alignedDNA data matrix, includingthe outgrouptaxa resultedin a total of 436 characters.of these characters,86 (2oo/o\were variable and 50 fll%) were phylogeneticallyinformative. Bootstrapanalysis ofthe aligned Reticulitermesspecies and the outgrouptaxa resultedin a consensustee (Fig. 2), (length= 132, Cl = 0.697,RI = 0.752), as documentedusing the Branch and Bound searchalgorithm of PAUP. The distinct clades from the maximum parsimony analysiswere: R. flavipes forming poorly supported sister group with R. tibialis, R.. hageni and R. virginicus. The distinctive relationship of these termite taxa has been observedrepeatedly in other genetic studies (Austin etal.2O02;Jenkins et al. 1998,1999). There was no haplotypeshucture observed amongthe R. tibialis andR. flavipes haplotypesin the presentstudy. C, E, G, L, J, T6 H, H1, ,fl E,G Hl, vl G,H E,J,H F,G E C,F,G,J FIG. L Distribttion of Reticulitermesspecies and haplotypes in Texas. DISCUSSION This study representsthe first attempt in over 16 years to update the current geographicdistribution and genetically categoize the genusReticulitermes in Texas. In the presentstudy, a clear bias associatedwith the frequencyofoccurrences from various Reticulitermes species which attack structures is reflected. The Eastem subterranean termiteR. flavipes is the predominantspecies observed in Texas.This is not surprisingas previoussurveys in Texasand other Gulf Coaststates similarly reflect this observation (Howell et al. 1983, Wang and Powell 2001, Messengeret al. 2002). However,with increasedurban expansioninto woodlandhabitats the occurrenceof other speciesmay be more frequently realized. Becauseof the limited numberof locationswhere samplesof R. hageni andR. virginicas usedin this study originatedfrom, little variation within these two specieswas detected. Rflnipeshap A RflovipeshapM RflavipeshapL RflnipeshapK Rflnipeshap J Rflavipeshapl RflnipeshapH Rflavipes hapG RflnipeshapF RflartipeshapE RflavipeshapD RflnipeshapC RflnipeshapB R tibialishapTl R tibialishapT3 R tibialishapT2 R tibialishapT4 R tibialishapTl R tibialishapT6 RtibialishapTT R virginicushap Yl R hagenihapHl H aureusSanta Rita AZ C formosanusGalveston Is FIG. 2. l63 singlemost parsimonious tree during a branchand 6ound search using PAUP*. Bootstrapvalues for 1,000replicates are listed above the branches supported at>SlYo Using the 165 rRNA gene,we have found that geneticdivergence can rangefrom as much as2.6% betweenR. virginicus andR. hageni to 9.2%obetween R. virginicus to R. tibialis (JWA. unpublisheddata). While less commonly encountered,R. tibialis was representedby sevendistinct haplotypes in this study.The fact thatTTo/oofthe R. tibialis populationsrepresent a unique haplotypeis intriguing andmerits further investigation. The lack of geographicpattems based on haplotypesobserved in R.flavipes is not surprising given the numerous opportunities for anthropogenicdisruptions. Some R. flavipes haplotypesappear to be unique or possibly conelatedwith geographyand warrant frntlrer investigil)on of Reticulitermesspecies in neighboringstates. By investigatingthe geneticvariation of Reticulitermesfrom larger geographiczones, the complex ecological demandsof this genuscan be better understood. Geneticvariation is important becauseit impacts on a species ability to respond to natural selection: selection is inversely proportional to genetic variation (Fisher 1958). While an individuals' fitness is determinedby interactionsbetween its phenotypeand the environment,social organisms' individual fitness is influencedby direct interactionsbetween phenotypes (Hochberg et al. 2003). The numerous haplotypes, without genetic stucture as often imposed by geographicisolation or distance,observed in the presentstudy might suggesta degreeof interactionthat has been observedin "open" (when termitesaccept alien homospecific individuals)termite populations.To more accuratelyassess this, intensivecollecting at various locations should be preformed and more robust statistical proceduresshould be applied. This phenomenonis variable, but has been demonstratedin ants (Forel 1920, Souli61960, Scherba 1964, Passera 1963, Benois 1972, Provost 1979) and occasionally in termiteswhere the degreeof aggressionbetween homospecific individuals may vary from one nest to another lNasutitermes corniger, Nasutitermes ephratae, Amiterrnes and Armitermes (Thorne .1982), Reticulitermessantonensis and Reticulitermesgrassei (Cl6ment 1978)]. For this reason,it is becoming increasingly important to evaluatethe genetic relationship between Reticulitermes so that a broader understandingof how sympatrichomospecific populations interact. This is importantbecause it allows us to betterunderstand the dynamic natureof controlling termiteswith newer control strategies (e.9.termite baiting regimes). It has been demonstratedthat Reticulitermescolony structwe and, therefore,gene flow can be more clearly understoodin North America and Europe when using mtDNA data(Austin etal.2002; Jenkinset aI.2001,2002;Marini and Mantovani2002). The Eastemsubterranean termite R. flavipes, incorrectly identified as R. santonensls,has been moved.about in Europe along trade corridors, expanding its known range tlroughout Franceand Europe (Laind 2002), Likewise in Texas, movementof R. flavipes is greatly influenced by trade corridors and partially explains the lack of haplotype structure observedin this studybased on geography.Colonies that mergeshared physical space and resoircesmay demonstrateno intercolonyagonistic behavior (Houseman et d. 2001). This lack of agonistic behavior between disjunct Reticulitermespopulations has been demonstratedextensively in Europe(Plateaux and Clement 1984, Clement 1986) and even in someinstances hybridization may be achieved(Clement 1977, 1979). The discontinuity of R. /lovipes in Texassuggests that fragmentationof Reticulitermespopulations due to anthropogenicdisturbances induces variations in their observedhaplotypes. Thirteen distinct hapolotypesfrom 68 different populationssuggests a considerable amount of genetic variation in this species,even without a geographic correlation as observedin other Rhinotermitidae (ALS unpublisheddata, Jenkins et al. 2002). More pronounceddifferences may yet be uncoveredin other endemicReticuliternes speciesin Texasas they are more frequentlycollected. Szalanski et al. (2003) have developeda quick and inexpensivemolecular method which can easily identi$ Reticulitermesspecies. The lack of diagnosticcastes for morphologicalspecies determination can be overcome employingmolecular diagnostic methods. Molecular methods also allow the utilizationof samplesalready in variouscollections can help facilitate more robust comparisons. ACKNOWLEDGMENT We would like to thank all contributorswho were willing to contribute specimens to this study,particularly the PMPs of Texas.Special thanks are given to Matt Messenger and Paul Baker for their contribution of samples.Research was supportedin part by the University of Arkansas,Arkansas Agricultural ExperimentStation. LITERATURECITED Anonymous.2003. NPMA webpagehttp://65 .217.229.17|lmedia/artgotect3our-home. asp. Austin, J. W., A. L. Szalanski,P. Uva, and A. Kence. 2002. A comparativegenetic analysis of the subtenanean termite genus Reticulitermes (Isoptera: Rhinotermitidae).Ann. Entomol.Soc' Amer. 9 5 : 753 -7 60. Benois, A. 1972. Etude experimentale de ]a fusion entre groupes chez la Fourmi Camponotusvagus, mettant en evidencela fermeturede la soci6t6.C. R. S. 274: 3364-3367. cl6ment, J. L. 1977.Caryotypes des Reticulitermes lucifugus Rossi. ch,romosomq 81: 169-175. Cl6ment,J.L.1978. L'aggressioninter et inffaspecifiquedes espdces frangaises du genre Reticulitermes.C. 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Entomol.96: l5l4-1519. Tajima, F., and M. Nei. 1984. Estimationof evolutionarydistance between nucleotide sequences.Mol. Biol. Evol. l:269-285. l0 Thome, B. L. 1982. Termite-termite interactions:workers as an agonistic caste.Psyche 89:133-l50. Wang, C. and J. Powell. 2001. Survey of termites in the delta experimental forest of Mississippi.Flor. Entomol. 84:222-226. 11 vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 INIYESTIGATION OF RED IMPORTED FIRE A}IT, ,SOI'MOPSIJ INWCTA. DAI\,TAGE TO PEA}.IUT,AMCHIS HWrcAEA. ForrestL. Mtchellt and Allen E. Knutson2 ABSTRACT Laboratorysnrdies dAernined that the red importedfue an\ Solerrysis irrictoBwq\ was unableto penetratepearnrt pods in orderto reachthe kernels. Kernelsremoved ftom the pods were acceptablefood sourcesand ants were sustainedover a 16-day period as long as accessto water was available. Ants were unableto draw enoughmoisture from the pods, kernels or artificial diet to sustainthemselves for more than a few days in the absenceof supplementalwater. Methoprenefire ant bait @xtingrrishil) reducedthe numberof foraging fire ants by 85-98% when appliedto two peanutfields. Howwer, there were no significant differencesin yield, grade or value of pearnrtsin plots wherefire ants nrmbersw€re reduced relative to untreatedplots. Rezultsof thesestudies indicated that red imported fire antgare unlikely to feed on and damagesound peanut podr. Damageto pods due to feedingby other insectsor cracksin the pod due to plant str$s (drought) may provide accessby red imported fire antswhich may then feed on the kem€ls. INTRODUCTION Red importedfire ant4 SolenopsisirwictaBven havebeen reported as serious pests of sweral crops. A numberof reportsdocument this speciesas a pest of the soybeanplant where it may feed on plantedseeds, genninating seeds and sedlings (Adamset al. 1983,Morrison et al. 1997, Smittle et d. 1983). Cor4 wlreat and sorghumseeds and seedlingsare also at risk from fire ant prdation (Dreeset al. 1991,Leonard et al. 1993,Morrison et al. 1997,Smittle et al. 1983). Both dry andsoaked seeds may be fed upo4 but soakedseeds sem preferred(Ihees etd. l99l). Damagetoseedsalsode,pendsontheoilcontentandthestateofdryrness.Fireants remove the €mbryo ofdry seedsofcor4 wheat and sorghumbut seldom eat the stsrchy endosperm@rees et al. 1991,Morrison et al. 1997). seedsofmore oily plaats(e.g. cotton and soybean)were cornpletelyconzumed (Monison a al. 1997). Plantroots arealso used as a food sourceby rcd importedfre ants. Althoughnot alwaysvisible, root damageinflicted by the ants is swere enoughto causestunting in soybean(Adams et al. 1983). Extensiveroot feedingon agronomicallyimportant plantswas reportedby Smittle et d. (1983). Ants, includingS. itwicta, havebeen associated with peanutin severalways. Doryline armyants arePod borers fiom SouthAfiica (Wrghtmanand Wightman 1994)tolndia andcasse direct feedingdanrage (IVlahto 1992, Singh and Singh 1992). Theseants are capableofboring ttuough the side of the pods and feedingon the kernels. Ants may also contibute to plam damageby herding and protecting honeyden,producing pest ins€cts (Scarborough1984, Weaving 1980),includfurg virus vectors zuchas aphidsand leaflropp€rs. rTexas Agrianltural ExperimentStatioq 1229N.Ifiglnvay 281, Stephenviile,T)I-7640| zTexas - CooperativeExt€nsio4 17360Coit Rd, Dallas, fX?'SZSZ 13 IntheUnited States,red importedfireants aremainlyconsideredbeneficialinthepeanut agrocecosystem (Kharboutli and Mack l9l) and feed on pest insects such as the lesser cornstalk bora, Ela,smopalpuslignosellus (Zeller) (Mack et al. 1988), and burrowing bug Pwrgaeusbilinealus (Say) (Smith andPitts 1974). Thereis no pubtshedrecord ofred imported fire antsfe€ding on peanutpods. Vogt et al. (2001)reported that pod damagewas less on plants growing withinfire antmoundsin OHahoma, and suggested that closeassociationwithhounds probably protects pods from feedingby wireworms and other pod feedinginsects. Even so, peanutproducts are well known to be attractiveto S. irwicta md baitscommonly include peanut oil as a component(Lofgen et al. 1963), where it can be more attrastive than traditiond attractants,such as molasses(Ali and Reagsn1986). Peanutmeal and peanutbtrtter are also attractiveto fire antsarid are also employed in baits(Hays and Arant 1960,Lofgren et d. 196l). During the growing seasonof 2000, peanutgrowers in ComancheCounty in central Texasobserved red imported fire antsfeeding on and damagingpeanuts pegs and pods. Ants wereassociated with pegsofpeanuts tlat wereactively growing, appearingto be eitherchewing or feedingon them. In one instance,ants were observedto be entcing into anddamaging pods of peanr.rtsthat were still aotivelygrowing. In addition, peanutplaots that had beendug and invertedto dry prior to thrashingwere ssento haveants actively feding on kernelsthrough damagedpods. Growers attributed loss not only to reducedyields, but lowered gradeson damagedkernels. Thereforegiven the attractivenessofprocessed peanut to antsand the ability ofants to feedon both seedsand subt€rranean roots, conc,emov€r whether red imported fre ant hasbecome a peanrt pestmay be valid. The following studieswere conductedto asc€rtainthe palatabilityof peanutpods and kernelsto red importedfire antsand to determinetheirimpact on peanutyield and gradein a field setting. MATERIALSAI{DMETHODS Assessingred importedfire ant damagein peanutwas donebotl in laboratoryand field settings. Two laboratoryexperiments were conductedto test whetherred importedfire ants could be inducedto feed on peanutpods or kernelsand to identirythe type offeeding damage doneto each. Methoprenefire art bait (ExtirgtdshT was appliedto two commercialpeannt fieldsin ComancheCounty, Ter(as, to suppressfire ant densitiesand to measurepeanut damage, yield and gradewith andwithout fire antspresent during the pod-filling stage. Red importedfire antsused in the laboratorystudy were collectedfrom activemounds on the groundsofthe Toras Agricultural ExperimeotStation in Stephenvi[g Tenas. Colonies were e)dractedby flotation and placedin plasticcontainers coatd on the inner zur&cewith a Fluonru barrier (Ilolleman and Elton 1965). Ants were fed a standardant diet (Bankset al. 1981,Drees and Ellison 1998) and removedftom the coloniesas neededfor the experiments. Coloniesand orperimentalcontainers were kept on a laboratorybench at room temperature. Peanutsused in the laboratorystudies were Tamrun96 collectedgreen at harvestfrom peanut fields on TAES property. Kemelsused in the orperimentswere shelledfrom thesepeanuts. ExperimentI consistedoffive treatmentsofants anddifferem combinations ofpeanut pods,ant dia and water ard two treatmentswithout antswhich serted as controls.(Table 1). Each replicationsused 50 ants, and treatmentswere replicatedfive times. Containersfor the e,:gerimentwere disposableplastic boxes measuring 16.5 cm2 and 4.5 cm deep. A 3-crn2hole was cut in the lid of eachcontainer and coveredwith fne cloth sealedin placewith hot glue to allow for air orchangeinto the container. Sidesofthe contaircr were costd with Fbonru to preventant escape.Cotton wicks saturatedwith water and ant diet were placedin smallpetri dishesin the bottomsof the containersand replenishedas needed.In treatmentswith peanuts, two podswere placed in eachcontainer, also in peri dishes.The orperimemlasted for ftre days. Antdietandwaterwereaddeddailyasnecessary.Peanutpodswereweighedatthebeginning T4 and end of the study and o TABLE l. Exoerime,ntl: Test of Red Imported Fire Ant AbiliW to Feedon PeanutPods. 2++-+ 3+++- 4++- 5+ 6-+- 7-+-+ Experiment2 was similarto the aboveer TABLE 2. Expuiment 2: Te.* ofRed Imported Fire Ant Abifity to Feedon PeamrtPods and Kernels. 2++ 3t-++ 4--T 5-++ 6-+++ l, the samecontainers were usedand treatmentswith pods receivedtwo pods per container. Treatmentswith kernelsreceived four kernelsper container. Water was replurisheddaily, but no peanutkern€ls or podswere addedor removedand none ofthe containersreceived ant di€t. Podsand kernelswere weighedat the beginningand end ofthe experimentand w€re oomined for signsof ant feedingdamage. Containers were examinedsix times during the snrdy,and an estimateof ant mortality, roundedto the nearest5olo, was madeon eachdate. To det€rrrine if red imported fre ants could causedamage to peanutpods in a field setting,nvo field studieswere conductedin ComancheCounty, T€xas, neaf, the shewhere the origlnal obseryationsof darnagewe're made. Two commercialfields of irrigatedpeanuts were eachdivided hto eight, 0.8l-hectare squareplots. In eachfield, four altern*ing plots w€re trestedwithmethoprenefre antbait (Extinguishru,Wellmark Imernaional)to suppressfireant numberswhile the rernainingfour plots in each field were left untreated as check plots. Methoprenefire ant bait was appliedonce on 8 June2001 at a rate of 1.68kilos per hectare usingaHerdseed€rmoutedb€hindafourwheelKarvasakiMuleru.Applicationwasmadeat 19.3 kph with a swathwidth of about 9.1 meters. The sandysoil and spreadingnature oftle peanutplurts madefinding fre ant mounds very diffiorlt in peanutfields. To overcomethis difrarlty, fre ant numberswere estimatedby trappingants in smallglass vials baitedwith candyand cat food. Prior to treatm€nt,four glass vials were wenly spaceddovm tlre c€nt€rof eachplot and placedon the ground eady on tlte 15 morningof25 May. Foragingfire antswhich discoveredthe bait recruitedoth€rworkers from nearbycolonies and beganremoving the bait. After one hour, the vials were picked up and capped,capturing worker ants inside. Bait vials were frozen and the numberof fire antswas recorded for each vial. Two montls after the bait application"fire ant activity was again monitoredon 2 August using 12 glassvials per plot to determinethe impactof the treatmenton the densf offoraging fire ants. A sampleof 10-12peanut plants was eachpulled from two locationsin eachplot on 6 September,and 100peanuts were collectedfrom eachsubsample (200 peanutsper plot). Each peanut pod was examinedfor the pres€nceofholes characteristicoffeeding by fire ants, wirewormsor other insects. Peanutswere machinehawested from an area six rows wide and 30.48 meterslong (0.0166 hectares)in the centerofeach plot on 4 Octoberand 29 Octoberin Field I and Field 2, respectively. HarvestweiSht, peroentmoisture, yiel4 and gradewere determinedfor each sample. Data on pod weight following exposureto fire antsin the laboratoryand weigh! yield and value of peanutsharvested from the field studieswere subjectedto ANOVA and means wereseparated by LSD test at 4 = 0.05. RESTJLTSAND DISCUSSION Both laboratorystudies demonstrated that redimported fire antswere unable able to feed on peanutpods and were certainlyunable to penetratethem. Ants died at tlre samerate when fed pods only as they did when o TABLE 3. PercentageAnt Mortdity Averagedover Five Repetitionsfor Each Treatmentin l. Treatment No 24 Pods+Diet+Water I 0 l0 l0 l8 Pods+ Water 2 2 l0 l0 l0 Pods+ Diet 3 l0 34 97 100 Pods 4 4 44 93 t00 Ants Only 5 2 82 100 100 Percentagemortality roundedto the nearestwhole number. Similarresults were achiwed in Experiment2 (Table4). In TreatmentsI and3, antshad accessto water and food sourcesofeither pods or kernels respectively. Tr€atments2 and4 were identical s(cept that water was excluded. Rates of mortality are again relatedto the presenceor absenceof water. Waterlesscontrols were conductedin an attemptto force ants to usemoisture from the food, gfeen podsor kemelsin orderto sustainthe,lnselves. Results of ant survivd in Treatments3 and 4 in ExperimentI (Table3) and Treatment2inEr 16 TABLE 4. PeroentageAlt Mortality Averagedover Five Repetitionsfor eachTreatment in Experiment2. Percerrtnce Mortalitv on Indicated Dava Treatment No. 136814l6 Pods'+Water I 0000s460 Pods 2 o 62 100 100 100 100 Kernels+ Water 3 00001222 Kernels 4 0 91 100 100 100 100 -Percentagemortalityroundedtothenearestwholerumber. Treatments6 andT in ExperimentI (Table l) and Treatme,nts5 and6 in Erpeimert2 (Table2) did not contain any antsin order that the changesin weight ofpods and kernelsdue to moistureloss could be comparedto lossesthat might be causedby antsscarifying the pod or kernel surfaces.No significantdiferences in pod weightsbetween treatments were observ€d in ExperimentI or 2 (Table 5). Treatment No. Experimut I Pods+Diet+Water I 3.9 Pods+ Water ) 4.0 Pods+Dia 3 4.6 Pods 4 4.4 Ants Only 5 No ants 6 4.4 No ants 7 3.6 Experiment2 Pods+ Water I 29.4 Pods 2 ,'-.0 Kemels+Wat€r 3 ttl a Kernels 4 ****b No ants ) 34.6 29.7b No ants 6 33.1 31.5b " Meansfollowed by the sameletter within a columnare not sigrrificantlydifferent; LSD, a= 0.05 u *r'*'r' .All antsin the experimentdied within 5 days. Kernels from Treatment3 had sma[, scallopedridges on the surfacemade by the mandiblesofindividual antswhich weretypical ofchewing insects(Fig. l). This sort ofdamAge could be expectedto occur on pods aswell ifthey hadbeen fed upon by the ants. Kernelslost an averageof 8.lolomore weight whenexposed to fire ants(Treatment 3) relativeto kernelsnot orposed(Table 5). Two monthsafter applicationofthe methopreneant bait, fire ant activity, as measurd in the baitedvials, was 85 and 98% lessin tle two shrdyfields relativeto tle untreatedcheck (Table 6). Howwer, there were no differencesin Vrel4 gradeor value W acrein either field (Table 7). Very little damageto pods was found in either peamrtfield. Only a smallfraction of the kernelswas accessibleto feedingvia compromisedpods, and the damageresembled that t'l FIG. l. Peanutkernel damagedby ant feedingin a laboratoryexperiment. FIG. 2. Tpical damagefound on peanutpods and kernelscollected from field trials. l8 TABLE 6. MeanNumber ofRed knported Fire Ants CollectedPer Vial on 2 August2000, Two Months after Aoolication ofM€thoDr€neBait. Percentage Field Check Bait Treated Reduction I 52 0.9 98 a 83 12 85 TABLE 7. AverageYiel4 Quality and Value of Peanrtsl{arvested from Plots Treatedwith MethooreneBait andNot TreatedFor Red Imported Fire fuits. Field Onea Field Twoa Treatment Grade Yield Value'/Acre yield Value/Acr€ 0bs/acre) 0bVacre) Extinguish 63.3 a 3,043a $843a 67.8a 3,M8 a $892a Check 59.8a 3,238a $839a 68.5a 3,543a $1,046a Meansfollowed by sameletter in a columnare not sigrificantly different;LSD, a = 0.05 TABLE 8. Mean Percentageof PeanutsWith Holes Throughthe Pod Resultingfrom Insect feeaing. Mean P€rc€ntaceofPeaouts with Holesa Field Check Extinguish I 0.6 0.8 ', 2.1 2.5 Peanutscollected 6 September,2001; 800 peanutsommined per treatmentin eachfield causedby less€rcornstalk borer andwireurorms (Table 8, Fig. 2). Redimported fire antsareknownto be seedfeeders, especiallyunder drought conditions nrch asoocrrred in the nortlsrtral Texasregion during 2000. Sorghum,corn, andcotton s€ed haveall beenreported zusceptibleto predatioq as haveunal seedlingsofotler crop species. Plantssuch as okra are commonlyattacked during the growing seasonbyred importedfire ants (Scarborough1984b Smittleet d. 1983).Howwer, theseare repeatable phenomena that have been observedmultiple years. In light of theseorperiments and information reportedin the scientificliterature on the feedinghabits offire ants,the wents of2000 in the peanutfields were likely due to a unuzualcombination of factors related to the prwailing drought conditions. Whether it will occur more commonly in the future is unknown" but the availability of a methoprenebait will allow concernedgrowers to apply minimallydisnrpive treatme,t for the red imported 6re ant infestingfields and field borders. Although methoprenefire ant bait significantlyreduced fire ant foragingin peanuts,further shrdiesare neded to demonstratean economicbenefit to treatment. t9 ACKNOWLEDGMENT The authorsthank JamesLasswell and Robert Whitney for assistancewith this project. SanfordPorter provided the FORMIS databaseused in the literature search. Supportwas providedby the TexasPeanut Producers Board and the TexasImported Fire Ant Researchand Management Project. LTIERATI.JRECITED Adams,C. T., W. A. Banks,C. S.Lofgren, B. J. Smittle,and D. P. Harlan.1983. ImFact ofthe red importedfire ant,Solenopsis itwicta Qlymenoptera:Fonnicidae), on the growthand yield ofsoybeans.J. Econ.Entomol. 76:1129 -1132. Ali, A. D., andT. E. Reagan.1986. Comparison ofbaits for monitoring foraging activity of the red imported fue ant (Hymenoptera:Fomricidae). J. Econ. Entomol. 79:1404-1405. Banks,W. A., C. S. Lofgerr, D. P. Jouvenaz,C. E. Stringer,P. M. Bishop,D. F. Williams, D. P. Wojci( andB. M. Glancey. I 981. Techniquesfor collecting,rearing andhandling importedfire ants. USDA, SEA' AATS-S-27,9pp. Drees,B. M., L. A. Berger,R. Cavazos,and S. B. Vinson. 1991.Factors affecting sorghum and corn seedpredation by foraging red imported fire ants (llyrrenoptera: Formicidae).J. Econ. Entomol. 84:285-289. Drees,B. M., andS. L. Ellison. 1998. Collectingand maintaining colonies ofred importedfire antsfor study. TexasAgricultural ExtensionService, Fire Ant Plan Fact Sheet#008, 2 pp. Hays, S. 8., and F. S. Arant. 1960. Insecticidal baits for control of the imported fire ant, Solenopsissaevissima richteri. J. Econ. Entomol. 53:I 88-191. Holleman, H. C., and E. T. G. Elton. 1965.Fluon baniers for confining non-flying insectsin opencontainen. Entomol. B€r. (Amsterdam)25:178-180. Kharboutli, M. S., and T. P. Mack. 1991. Relativp and seasonalabundance of predaceous arthropodsin Alabama peanut fields as indexed by pitfall taps. J. Econ. Entomol. 84:1015-1023. konard, B. R., P. A. Clay, and T. J. Riley. 1993. The red imported fire ant: a pest of grain sorghumin reducedtillage productionsystems in Louisiana.Louisiana Agric.36:16. lofgerU C. S., F. J. Bartlett, and C. E. SEinger.1961 . Importd fire ant toxic bait studies:The evaluationof variousfood m*erials. J. Econ.Entomol. 54:1096-1100. Lofgren, C. S., F. J. Bartlett, and C. E. Stringer. 19f1. Imported fire ant toxic bait studies: Evaluationof caniers for oil baits. J. Econ. Entomol. 56:62-66. Maclq T. P., A. G. Appel, C. B. Backman,and P. J. Trichilo. 1988.Water relations of several arthropodpredators in the peanutagroecosystem. Environ. Entomol. 17:778-781. Mahto, Y. 1992. Varietal susceptibility of Dorylus orientalis Westwood (Hymenoptera: Formicidae)in groundnutvarieties. J. Entomol.Res. (New Delhi) l5:144-148. Monison, J. E., D. F. Williams, D. H. Oi, and K. N. Potter.1997. Damage to dry crop seedby red imported fire ant (Hymenoptera:Formicidae). J. Econ. Entomol. 90:218-222. Scarboroug[ T. A. 1984.Mutualisrnoftheredimported firent"Solenopsis fzvictaBurcn,with honeydew-producingHomoptera. Ph.D. dissertation., Texas A&M University,L23 pp. Singh,T. V. K., and K. M. Singh. l992.Etreotof different intercropson termitesand oriental army ant, Dorylus orientalis Westwooddamage to groundnut.Indian J. Plant Prot. 2A:129-132. Smith, J. W. Jr., and J. T. Pitts. 1974.Pest stafis of Pangaeusbilineatus attackingpeanuts in Texas.J. Econ.Entomol. 67 :lll-113. 20 Smittle.B. J., C. T. Adams,and C. S. Lofgren.1983. Red importedfire ants:Detection of feedingon com, okra andsoybeans with radioisotopes.J. GeorgiaEntomol. Soc. l8:78' 82. vogt, J. T., R. A. Grantharn,w. A. smitlu and D. c. Amold. 2001. Prey of the red imported fire ant (hymenoptera:Formicidae) in Oklatromapeanuts. Environ. Entomol. 123'128. Weaving,A. J. S. 1980.Observatiorc on Hilda patrzelis Stal. (Homoptera:Tettigometridae) and its infestation of the groundnut crop in Rhodesia.J. Entomol. soc. South. Afr. 43:15l-157. wightrnaru J. A., and A. s. wighunan. 1994.An insect,agronomic and sociologicalsurvey of groundnutfields in southemAfrica. Agr. Ecosyst.Environ. 5 I :3I l -33I ' 2T vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 EVALUATION OF COTTON STALKDESTRUCTIONFOR CONTROL OF PINK BOLLWORM OEPIDOPTERA GELECHIIDAE) Eric T. Narwich Robert T. Statenr,and StephenL. Birdsall 2 University of California CooperativeExtension, University of CaliforniaDesert Research and ExtensionCenter, 1050East Holton Road,Holwillg CL9225O ABSTRACT Threecotton stalkdestnrctionmethodswereevaluated fortheirimpact onpinkbollwornl Pectinophora gosslpiella (Saunder$, larval mortality. Cotton stalks were shreddedusing a rotary shredder,a flail shredder,and a forageharvester. The rotary andflail shreddersleft cotton bolls and shreddedstalks in the field. The foragehaf,vester removd much ofthe cotton residue from the field. The stalk shreddingand harvestingtreatments were evaluatedfor destructionof cotton bolls andpink bollworm larvae.Numbers of intact greenbolls, intact dry brown bolls, and surviving pink bollworm larvae were similar for the three treatments. Pink bollworm overwinteringsurvival lwels in the field were similarfor all stalk destructiontreatments. Cotton plantresiduewasremovedfromthefield witla forageharvesterand evaluated forpinkbollwonn overwinteringzurvival. Pink bollworm mortality exceeded99.9 Yoin shreddedstalks from the forage hanrestef,treaunent. INTRODUCTION The pink bollworrl Pectinophora gossypiella (Saunders),is a major pest of ootton Gossypiamspp. in the southwesternUnited States,causing yield lossesas high as 53%in upland cotton G. hirsatum$lewteberry et al. 1977).Since the establishmentofthis pestin Arizona and Catifomia the impaot on the cost of cotton production has been dramatio. Prior to the introduction of Bollgard@(Monsanto Company,St. Louis, Ilfissouri) transgeniccotton with Bacillas tlruringiensis slbsp. karstaki @.t.k) CrylAc protein, pink bollworm was tte most dam4gingcottonpestin SouthernCalifomia and Arizona and continuestobeamajorpestinnon- 8/ cottonvarieties. During the years1 966- I 9E0there was a generaltrend ofincreasedproduction costs due to pink bollworm control and secondarypest problems, and a generaltrend of decreasedyield in lint and seedin the Impoial Valley of Califomia @urrows et al. 1982). The pink bollworm is best adaptedto af,easwith low rainfall and a long growing season. Developmentfrom eggto adult requiresabout 25 to 30 daysin midsummer.Population densities reachmocimum lwels in August and Septembercoresponding to the fourth or fifth generations. Shorteningdaylenghafterl5 September(13horless) stimulatesfullygrownfourth-instarlarvae to spin a light oocoonand enter diapause(Adkisson 1965).Larvae in diapausepass the winter in seeds,old bolls, and trash in the fields or at gins and seed-storagefacilities (Noble 1969). Overwimeringlarvaepupateinearly spring and emergeasmotlsinlate springand early summer to infest the new crop ofcotton (Ilermeberry1986). I USDA APHIS, 4125E. Broadway,Phoenix, AZ 85240 2 AgriculturalCommissioner, l5O S. 9d'Street,El Centro,CA92243 23 Pink bollwormlends itselfto dispersalby man.Commercial transport of cotton seedrapidly facilitatedthe invasionof pink bollworm to most major cotton-producingcountries throughout the world. Pink bollworm becamea widespreadpest of commercial6tton in Arizona and California after it was brought into the areain infestedcotton seed.Some larvae €Nrter cotton seed,diapause and overwinter.Pink bolhvormis movedwith cotton seed,baled lint, mechanical cotton pickers, and vehiclesused to transport seedcotton, cotton seed,oil-mill products,and other items subjectto contaminationby infestedcotton seeds(Noble 1969). Control of pink bollworms in cotton seedis necessaryfor managementin a generally infestedarea. Quarantine regulations require infested materials to be treatedto renderthem free oflive larvae before they are moved out ofthe infested area. Soon after the insect became establishedin this country,federal and state goveflments adopted regulations requiring that grnS and oil mills in quarantined areasbe providedwith equipmentfor killing the larvaelNoUte t eOe;. Pink bollworm is not known to be permanantlyestablished in the San JoaquinValley, llthough adults are apparentlycarried there by winds from SouthernCalifornia (Stern and Sevacherian1978). Smallnumbers oflarvae are found in tlre valley occasionally.Timely plow down in compliancewith local regulationshelp prwents wider infestation.Release of millions of sterilized adults each seasonby the USDA in cooperationwith other public agenciesis designcdto prev€ntimmigrating adults from matingand reproducing(Anonymous 19g4). Practicalcontrol ofthe insectstill is dependenton cultural practices,,Bt transgenic cottoq andinsecticides. Mandatory cultural control zonesare in effectin Arizon4 Californi4 andcertain regulatedareas in Louisiana(Noble 1969).Cotton fruiting terminationon or beforeI September, shreddingofcotton stalks,and double discing ofthe crop residueby I Novemberwas mandated for the ImperialValley by CalifomiaDepartment ofFood andAgriculture regulationsfrom 1989 through1999. Cotton stalk shreddingusing a foragehawester provided great er tttangg9 yokill ofnative pink bollworm larvaein a studyby Natwick and Staten(1987) in cotton bolls removedfrom the field with shreddedstalks. The study did not addressthe overwintering emergenceof pink bollworm motls from lawae survivingin the soil or in bolls left in the field. Standardpructices for shreddingof cotton stalksfollowing lint harvesthas been accomplished utilizing eitier a flail shredderor rotaf,y shredder(Chapman et al. lg6l,Watson et al. 1970).Interest in the for4ge harvesterforcotton stalkremovalwas orpressedby companiesinterestedinburningthismaterial to generateelectricity The stalk piles ofcotton biomasscould be a potential sourceofpink bollworm overwinteringin the centralvalley of California. With the energycrisis in Californi4 thereis renewedinterest in usageof crop residuesfor biomassas fuel for generatingelectricrty. The resultsof this study,thouglr l5 yearJoH, could be usedto help dwelop guidelinesto preventthe hartor4geor spreadofpink bollworm throughthe mov€tnentand storageof shreddedcotton stalksto be usedas a biomassfuel. Pinlcbollwormhas been under an eradicationprogram inthe oentralvalleyofCalifornia for over 36 years.Eradication of this pest was unsuccessfullyattempted in the Imperial Valley, Californiaduring the 1990's.An eradicationeffort is plannedfor the southernCalifornia valleys beginningin 2006. Eradicationmay be successfulin the future, but we must be preparedfor continuedmanag€ment of pink bollworm until eradicationis successfi.rl. The objectiveofthis studywas to comparea foragehawester, used to hanest cotton stalks for biomasselectrical generatio4 to a flail shredder and a rotary shredders,the standard equipmentused for stalk destructionto oontrol pink bollworm management,thereby verifying that the forageharvester can provide a level ofpink bollworm larval mortality equivalentto that of the standardequipment. 24 MATERIALS AND METHODS Lantal Poprlation. Cotton plantedat the University of CaliforniaDesert Researchand ExtensionCenter was harvestedfor lint l0 November 1988.Ten thousandfive hundredgreen unopenedcotton bolls were extracted from stalks in a 3.25 ha and a 0.4 ha block of cotton immediatelyfollowing lint harvest.Sets of 50 bolls eachwereplaced into 84 vmtilated pl-astic incubationboxes and held outdoorsin a shadedarea. The 84 incubationboxes were dividedinto setsof 2l boxesfor examinationl, 8, 15, or 22 daysafter lint harvest. Numbersof pink bollwormlarvaethatemerged and droppedto thebottom ofthe boxeswerereoorded,afterwhioh bolls were dissectedto determinethe numbersof small (first or secondinstar), medium(third instar), and large (fourth instar) larvae remainingwithin the setsof 50 bolls for eachset of 2l boxesduring the aforenrentionedpost-harvest sampling periods. Numbers ofpink bollwormsthat were found within dissectedbolls alongwith larvaethat cutout ofbolls were recordedfor eaoh box for eachpost-harvest time period. To determinethe infield pink bollworm populationimmediately post-hanest, and prior to shredding,1,500 cotton bolls were openedand examined for larvae.Stand counts were estimated by countingthe numbersof cotton plantsin 4 m of row from ten locationsin the field. The mean numberof pink bollworm per boll, meannumber of bolls per plant, and meannumber of plants per ha were usedto calculatetlrc numberof pink bollworm per ha. ShredderEvalaation. Three cotton stalk shreddingtreatments were replicated6 timesin a randomizedcomplete block designin a portion of a 3.25 ha block of cotton. The three shreddingtreatments inoluded a JohnDeere model 3960 forage hawester, which removedcotton stalkswith bolls, a Caldwellmodel S-7 rotary shredder,and a Dandall76 RC flail shredder.The flail and rotary shreddersleft the shreddedstalks and bolls in the plots. The plot sizewas 8.2 m by 30.5mwith 2.lm bufferzones between plots. Immediately following shredding,intact green and dried unopenedbrown bolls were collectedfiom two areasof soil surfacemeasuring 0.75m2 in eachplot. After recordingthe numbersof green and brown bolls from each plot, the cotton bolls were crackedopen and numbersof live pink bollworm larvaewere extractedand their nurnberswere recorded. The plots were discedtwice to approximately10.20cm to 15.25cm"which is the standard"plow down" practicefor ImperialCounty cotton. Four pink bollworm emergencecages, I m2at the base,were placedin eachplot and remaineduntil 10 July 1989.Emergence cages were ercminedfor pink bollworm moth emerge,ncefrom 13 December1988 through 9 July 1989.The emergencecages were checkedonce weeHy from 5 March through 4 April, then twice weekly until 9 July 1989. OverwirxeringIn ShreddedCotton,Stalfr.An experimentwas implemented to measurethe overwinteringsurvival of pink bollworm in shreddedcotton stalks.The 3 treatme,nt,12 replicate, randomizedcompleteblockdesignexperimentutilized lm2basepinkbollworm emergencecages. Emergencecages were placed on soil which had been fallow for more than a year prior to placementofthe c4ges.The three treatments were 0-064m3 ofshredded cotton stalks(equivalent to stalks from 0.007771ha containingapproximately 545 pink bollworm larvae),400 green unopenedcotton bolls averaging1.36 pink bollworm larvaeper boll (approximately544 pink bollworm larvae),and bare ground. The 400 greenunopened cotton bolls were coveredwith a layer of straw to simulatethe insulatedconditions available to pink bollworm larvae in the shreddedcotton stalk material. Emergencecages were checkedfor pink bollworm moth emergenceon the same dates as emergenc€cages placed in the cotton shreddermethods experimentprwiously described. Six walk-in cages,2m tall by 4m wide by 8m long, wereerected to measurepink bollworm overwinteringsurvival in large piles of stneddedcotton stalk material.Three cages each were erectedover 25m3of shreddedcotton stalk material and tlree cageswere erectedover bare ground. Within eachwalk-in oage,a stakewas placed in the middleofthe interior space.Affxed 25 to eachstake were deltasticky traps baited with a gossypluresepturq a lure with a syntheticpink bollworm pheromone( I : I Z,Z:2,E7 ,ll-hexadeoadienylacetate) used as a sexattractant for male moths.Six stakeseach with gossyplurebaited delta traps were alsoplaced around the perimeter of the areacontaining the six walk-in cages.All delta traps were checkedfor pink bollworm mothsfrom 25 March through6 July 1989. Red dyemarked pink bollworm pupaewere received fromtheUSDAAPHISrearing facility inPhoenix, AZ,weekly. Twenty pupae (ten males and ten females)wereplacedin eachofsix cardboardtubsweeHy; moths were released into eaohwalk-in cage as they emerged.Numbers of native and marked moths captured in delta traps w€f,e recordedboth within and outsidethe walk-in cages. Metabolic Heating.Tlrcfinocoupleprobes were insertedat various depthsinto shredded cotton stalkmaterial piled at the endofthe 3.25 ha cotton block. All probeswere inserted0.33m abovethe soil surfaoe.Two probeseach were inserted 0.33m, 0.66rq LOm,and 1.33minto the shreddedcotton stalkmaterial pile to monitortemperature from microbialdegradation. One probe at eachinsertion depth was located on the west andeastside ofthe pile, respectively.Probes were insertedon 18 Novemb€rand removed 10 December.Probes were connectedto thermographs for continualrecording oftemperature changes. A glassbulb mercurytlermometer wasused 19 Novemberand periodicallythereafter to verifr the nccuraoyoftle thermographreadings. ShreddingMornlif. Shreddedcotton stalk samplesfrom the foragehawester were fed througha gin trashmachine in 0. I 3 m3increments to extractpink bollworm larvae.The effcienoy ofthe gin trash machineto r@over pink bollworm larvaewas checkedperiodically by releasing 20 markedlarvae, reared on red dye mediurq into 0.13m3of shreddedcotton stalk rnaterialand then feed into the gin trashmachine. A total of340 markedlarvae were fed throughthe gin trash machinein 2.2lnf of shreddedootton stalks. The gin trash machinewas utilized from 23 Novernbertlrough12December 1988. Atotal of9.2m3of shreddedcottonstalkmaterialfrom the forageharvester was fed through the gin trash machine. Stalk Pile Sanitation. Prior to the I January 1989 "plow down" date, all non-caged shreddedcotton stalk materialwas removedand burned. The areapreviously under the shredded cotton stalk materialpile was then coveredwith 26 pink bollworm emergencecages, 3m2 at the base. Thesecages were sampledon the sameschedule as lm2 cagesused in the in-field shredder evaluationstudy. Pink bollworm larvaand moth datawero analyzedusing A}.[OVA. Whentreatment effects were significant, means w€f,e separatedusing the least significant difference (LSD) test (Anonymous1989). RESULTS Larval Population. The infield pink bollworm populationimmediately post-harvest was estimatedto be7o,l46lawae per ha. The field plant populationcalculated to be 47,443plants per h4 averaging1.085 unopened cotton bolls per plant and 13627 pink bollworm lanraeper boll. Cottonbollsinincubationboxesyieldedasignificantly (P<0.05) decreasingnumberofsmall (secondinstar) andmedium (third instar)larvae within the bolls with increasingnumb€rs of days after lirt harvestfrom I dayto 22 days(Tablel). While smalland medium larval numberswithin bolls were decreasingover time, the number of large (fourth instar) lawae was increasing signifioantly(P<0.05) over time as was the numberof larvaeemerging from bolls. Thesedata indicatethatsmall andmediumpinkbollwormlarvae continueto feed andmatureto becomelarge larvaewhich orit the greenbolls over a periodofa few daysto severaldays after lint harvest.Our resultsare in agreementwith by prwious researchby Henneberry(1986) in that it is beneficial in a managementscheme to minimizepink bollworm overwinteringand to shredthe cotton stalks and bolls as soon as possiblefollowing lint harvest. 26 TABLE l. Mean Small, Medium, and Large Pink Bollworm larr"ae Wi6in or Emerged From Groen CdonBotts for%nous DaF Afi€rFb st, Days Mean Snall Larvae" Mean MediunLrvae" Mean Larqe Larvae' a$er Harvest Within Energed Wiftin E r€rged Wi6in Eneryed I 15.6+2.3 a 0.00+ 0.00b 13.9+ 1.9a 0.05+ 0.05b 42.4+2.2b 1.0+ 0.4d 8 7.0+l.3b 0.19+0.15ab 8.8+ 1.8b 0.14+ 0.08b 55.6+ 7.1ab 8.5+ 0.8c l5 4.0+ 0.6bc 0.05+ 0.05b 6.0+ 0.7bo 0.05+ 0.05b 62.1+3.7a ll.9+ l.2b 22 2.5+0.6c O52i0.21a 2.8+ 0.5c 0.57f0.19 a 54.3+ 5.5ab 15.2+1.6a " Moans within a colunm followed by tho same lerfieraro not sigrificantly differsrt (ANOVA LSD, P<0.05). Small larrrae= Secondinstar, medium larvae = third instar, large larrrae= frurth instar. ShredderEvaluatioz. Following cotton stalk destructioq soil surfaceareas of 0.75m2in eachplot yieldedan averageof5.2 unope,nedgreenbolls and 33.2 unopenedbrown bolls forthe rotary shreddertreatment, 2.3 unopenedgreen and 48.2 unopenedbrown bolls for the flail shreddertreatment, and 3.2 unopenedgreen and 45.2 unopenedbrown bolls for tlre forage harvestertreatment. The greenand brown boll countsfor the aforernentionedtreafinents were not significantly different using AI.IOVA (P = 0.05) (Table 2). The mean numbersof pink bollworm from the bolls collectedfrom 0.75m2areas were 14 and ll for the rotary and flail shredders,respectively, and I 1.5 for tle forage harvester;there were no significantdiferences amongthe treatm€nts. Thereforg basedon the data presented,the forage harvestenneither provided an advantagenor a disadvantageas comparedto the traditional shreddingpractices. TABLE 2. Mean Pink Bollworm Larvae within Cotton Bolls, Mean GreenUnopened Cotton Bolls, andMean Brown UnopenedCotton Bolls Remainingon the Soil surfacefollowing Various ShreddinsTreatments. Holwille. CA. 1988. Treatmerf I.arvael0.75m2^ GreenbollV0.75m2" Brownbolldo.T5m2o Rotary shredde,r 14.0+ 3.8 5.2+1.4 33.2+3.7 +0.4 Flail shredder I 1.0+ 4.5 2.3 48.2*8.3 + Forageharvester ll.5 * 2.8 3.2 0.8 45.2*6.3 'There were no significantdifferences among means within columns(Al'[OVd F0.05). Themean numbers for overwinteringpink bollworm mothsemerging from rotary shredder, flail stnedder, and forage harvester treafinents after plots were double disced were not significantlydifferent (P : 0.05) (Table 3). Data indicatethat the forageharvester is equivalent to the standardstalk destruction equipment for destructionofpinkbollwormlarvae andtherefore could be usedas an alternativeto use ofa flail or rotary shredder. 27 TABLE 3. MeanPink Bollworm Moth Emergenceinto Large CagesPlaced in the Field Various 1989. Treatment Rotary shredder 14.7+3.1 Flail shredder 18.8+ 2.6 Forageharvester 21.2+3.1 " Therewere no significantdifferences (AlfOVAs ^F0.05). OverwinteringIn ShreddedCottonStallcs. Overwintering pinkbollworm meannumbers of moths from small emergencecages for shreddedcotton stalks (0.25), out of an estimated populationof 561 pinkbollworm larvaeper 0.008ha at harvest,was significantlyless (P < 0.05) comparedtothemeanfromthe400unopenedgreencottonbolltreatment(17.33)(Table4).The bare ground treatment did not yield any pink bollworm moths. Percentageoverwintering emergencefrom green bolls was 3.186, which is similar to emef,gencefrom other studies (Natwick 1986, Slosserand Watson l972).Pacerfiage overwinteringemergence from shredded cotton stalkswas 0.0446,which was 7l fold lower than the emergencefrom greenbolls. TABLE 4. Mean Pink Bollworm Moth Emergenoefrom Cotton Crop ResidueChopped with a ForaceHarvester. Green Bolls. and Bare Ground.Holtville, CA. 1989. Treatment Mean numbersof pink bollworm moths' +3.82 Greencotton bolls 17.33 a + Cotton crop residue 0.25 0.13b + Bare ground 0.00 0.00b " Memsfollowed by tho sameletter are not sipificantly difr€r€ilt(ANOVA, LSD, P<0.05). Both native and markedpink bollworm mothswere capturedin gossyplurebaited delta stickytraps within the threewalk-in cageswith shreddedcotton stalkmaterial. Only marked pink bollworm mothswere capturedfrom three walk-in cagesover bareground. The six deltatraps outsideofand encirolingthewalk-incagestrapped24mtiveard2 marked pinkbollworm moths. Theneed for monitoringwith gossyplurebaited traps placed around stalk piles ofshredded cotton stalksstored for biomasselectrical generation was demonstratedfrom the resultsofthis study. Metabolic Heating.Internaltemperature ofthe shreddedcotton stalkpile at lm and 1.33m probeinsertion depths exceeded 66"C I day after shreddingand averaged over 38"C for 19 days dueto microbialmAabolic heating. Temperatures at 0.33mand 0.66 minsertiondepths exceeded 38"C I day after shreddingand exceeded55'C 3 daysafter shredding.From the 5th to the fth day after shredding,a strong cold northwestwind cooled the stalk piles to below 38"C on the west side while the east side averagedover 38oCover all depthsofinsertion during the same period.Piles ofshreddedcotton stalksheated sufEciently to kill pink bollworm larvaeunable to move quicHy to the pile surfaceor into cool soil. Pink bollworm larvae will die if exPosedto temperaturesof 60"C for more than 3 tL and high mortality ratesoocur at 55"C for I h or 67%;o to 99/o mortality at 50"C for 8 h or more (Chu 1988). Partial or completepink bollworm 28 reproductivesterility is inducedfrom heatingin q DISCUSSION Severalplants have been built in Californiafor burningbiomass to generateelectricity. A concernfrom the cottonindustryhasbeenthemovementofoottonbiomass from sbreddedstalks asa potentialsource of overwinteringfor pink bollworm. Resultsfrom our researchsuggest that there is minimal risk of harboringoverwintering pink bollworm larvaewhen cotton biomassis harvestedusing a forage harvesterdue to the 99.9 yo mortality of larvaefrom the shreddingof stalks and bolls. Further the microbial metabolicheating in piles ofshredded cotton stalks is capableof killing pink bollworm lanae or causingserrual sterility. Pink bollworm moth captureresults from our studyare in agreementwith previousresearoh resultsindicating a needfor gronitoring of pink bollworm moths,using gossyplurebaited live traps or delta sticky traps, in the vicinity oflarge stalk piles ofshreddedcotton stalks(Natwick andStaten 1987). LITERATURE CITED Adkisso4P. O. 1965.Biological Clocks and Insect Photoperiodism. Tex. Ag. Agr. Exp. Sta. Agron.Notes, Dec. 3l:31-36. Anonymous. 1989. MSTAT-C. A microcomputerprogram for the desigq management,and analysisof agronomicresearch experiments. Mchigan StateUniversity, East Lansing, Mch. Anonymous1984. Integrated pest manag€rnentfor cotton in the westernregion of the United Stateg Univ. Calif , Div. of fur. andNatural Resoure,esPubl. 3305, lzt4 p. Burrows,T.M.,V. Swacheria4H. Browning,and J. Baritelle.l982.The history and oost ofthe pink bollwormin the ImperialValley. Bul. Entomol.Soc. Amer. 28:286-290. Chapman,A. J., O. T. Robertson,and L. W. Noble. 1961.Evaluation of stalk shreddersand cuttersfor pink bollworm control, J. Econ. Entomol. 54l-791-92. Chu, C. C. 1988.Effect ofboll tntperature on larval mortality ofpinkbollworm Pectinophora gossypie I Ia (Saunders).Southwestem Entomol. I I : I 85-I 89. Fye, R. 8., and H. K. Poole. 1971.Effect of high temperatureson fecundityand fertility of six Lepidopterouspests of cottonin Arizona.U. S. Dept. Agr. Prod.Res. Rpt. 13l. Henneberry,T. J. 1986.Pink bollworm manag€mentin cotton in southwesternUdted States. U. S.Dept. Agr., Agr.Res. Serv., ARS-51,45 p. Henneberry,T.J., L.A. Bariola"K.E. Fry, andD.L. Kittock. 1977.Pink bollworminfestations and relationshipsto cotton yield in Arizona. U. S. Dept. Agr. ARS W-49. 29 Natwick, E. T. 1986.Crop rotationand pink bollworrr in the emergence.pp. 74-Be,In T. Kerby, (ed) CaliforniaCotton ProgressReport, Univ. Calif. Coop. Ext. Agr. Expt. Sta. Publ. Natwiclg E. T., R. T. Staten.1987. Destnrction of pink bollworm: Feasibilityofusing a for4ge hawesterto finely chop cotton stalks and bolls for safe storage.pp.262-267 , In I . M. Brown (ed)Proc. Beltwide Cotton Prod. Res. Conf., 1987. Noble L. W. 1969.Fifty years of researchon the pink bollworm in the United States.U. S. Dept. Agr. Hdbk. 357,62 p. Slosser,J. E., T. F. Watson.1972. Influence of irrigationon overwintersurvival ofthe pink bollworm.Env. Entomol.l:572-76. St€rq V. , andV. Sevacherian.I 978 . Long-rangedispersal ofpink bollworm into the Sanfoaquin Valley.Calif. Agr. 32: 4-5. Watson,T. F., W. E. Larso4 K. K. Barnes,andFullerton. 1970. Value of stalk shreddersin pink bollwormcontrol. J. Econ.Entomol. 63:1226-28. 30 vol".29 NO.1 SOUTHWESTERNENTOMOLOGIST MAR.2004 SURVTVAL OF BLACK CUTWORM'LARVAEA}TD OBSERVATIONS ON LARVAL PREDATION BY TIIEIF ANf IN A TI.]PJGRASSECOSYSTEM T. A. Royer andN. R. Walker Deparfinentof Entomologyand Plant Pathology 127Noble ReseanchCenter OklalromaState University, Stillwater, OK 7 407I ABSTRACT Studieswere conductedduring the sumrnerof 2002 to evaluatesurvival of artificially- infested black cutworm Agrotis ipsilon (Hufingel) larvae placed in cageson a bentgrass green. Plots were establishedon a site containing creeping bentgrass,an endemic population ofthief ants, and a gradient of shade. Influence of shade(firll, partial, and abse,nt),timing of cutworm release (night and day) and type of barrier system (cages placed dfuectly on the turf suface, cagesplaced into a ring of shaving cream, or cages placedinto a ring ofshaving creamwith an inner plastic cageplaced into the soil surface) were evaluatedfor their effect on black cutworm survivd. Survival of cutwomr larvaewas affectedby predationactivity of the thief ant Solenopsismolesta (Say), which ulas able to prey on larvae that were confined within the cages. Shadeindirectly influenced cutworm surr"ival becausepredation activity by S. molesta appearcdto be less intense in shade compaledto full su. Cagesplaced in the most heavily shadedcages contained an average of 4.8 live larvae per cage. No larvae confined in cagesplaced in full sulight survived and their physical remains were completely removed by ants. The time at which larvae were releaseddid not affect larval survival. The tlpe of balrier system used had a significant effect on cutworm sunival. The most effective barrier system for excluding antscontained an additional buried plastic barrier and resultedin highestcutworm survival. The results of these studies suggest that black cutwonn larvae survive better wtren confined in cagesthat are desigred to excludeS. molestathrough subtenaneanmovement and that S. molestamay ptovide effective natural control of cutwormsin golf greens. INTRODUCTION The lawal stagesof the black cutwonn, Agrotis ipsilon (Httfnzgel), bronzedcutwoml, Nephelodesminians (Guenee),and variegatedcutworm, Peridroma sazcla (Hiibner), are destnrctivepests on numerouscrops including turfgrassesin the southernUnited States. While they can be desEuctiveto highly managedturf, most of the drmrge occurs from feeding by the penultimate and ultimate instars, which consumelrge amountsof plant material in a relatively short period of time. In additiorl they are susceptibleto predation Noctuiidae 'Hymenoptera:ilepidoptera: Fonnicidae 3l by vertebratepredators whose feeding activities causecollateral damageto turf. On the other hand early instar cutwonn larvae causenegligible or minor damage,and are more likely to be preyedupon by arthropodszuch as carabidand saphylinid beetles,spiders, and antswhich causelittle or no collateral injury from their feedingactivities (Lopez and Pott€r 200,0,Znngand cibb 2001). Management systems for maintaining hrrfgrass often rely heavily on pesticide applications to suppr€ssinsect, disease,and weed pests (Anonymous 1999). Broad sp€ctur& synthetic insecticides are often used to rnanage cutwonns. Due to the cutworms' nocturnal feeding habits, insecticide applications are frequently initiated to control cutworms after extensive damage has already occurred or in responseto a perceivedthreat of infestation. Frequentuse of such insecticidessigpificantly affectsnon- target or beneficial ins€ctsthat dwell within or adjacentto the treatedarea (Cockfield and Potter1983, 1985; Kunkel et al. 1999;Z,enget and Gibb 2001). Recently, there has been a need identified for more rpsearcheffort on the biological contol ofgreen industry pests(Carnpbell et al. 2001). Ants are commoninhabitants ofthe urban landscapeand can be an important arthropod predator in the turfgrass ecosystem (Hayes l920,Lopezand Potter 2OOO,Znrryerand Gibb,2001). Ants also enhancenutrient recycling, mix organic and inorganic componentsof the soil environmen! and preventthe developmentof soil horizons @axter and Hole l967,Warg st al. 1995). Despite their potentially beneficial activities, ants arc often perceivedas a nuisancedue to their mound building behavior and becausethey may directly or indirectly causethe thinning of a turfgrassstand around their nests(Potter 1998). The objectives of this researchwere to assesseffects of shade,diel periodicity, and exolusion stategies on lanral survival and predation intensity by the thief ant, Solenopsis molesta(Say), on black cutworm larvaeconfined in cageson a golfcourse green. MATERIALS AND METHODS Field plots were establishedin the summerof 2002 at the OklahomaState University, Departnent of Entomology and Plant Pathology ResearchFamr located in Stillwater, Oklalroma. T\e 223-r* rectangular study green consistedof two-year old, established creepingbentgrass, lgrostis palustris Hudsoncv. SR-1020(Seed Research ofOregon Inc., Corvalis, OR), grown on a 3Ocm-deepsand based root zone. The north side of the green was lined with a row of tr,ees,Umus pamifolia, 15-18 m in height, while the south side was free ofvegetation. The trees createda shadegradient on the green that progressed from north (heavy shade)to south (no shade). A subsurfacedrainage system, consisting of gravel ( JZ Cages, similar to those described by Heller and Walker 2002, wete constnrcted of poiyvinylchloride plastic and "ouo.d at one end with nylon window screening(lcm2) secrred-usinghot-melt glue. Each cage measured30-cm diametet x l5-cm in height a1d weighed approximately 1,3559. The edgeof the cageexerted a force of 13.79lcm'on Oe turfgass "-opy. Black cutw^ormlarvae (secondand third instars)urcre prwided by Dow Agisciences una stotea at 4oC prior to release. Larvae were countedand trans rred to breathablepaper containers for releaseinto the cages. Cageswere arrangedon the turf in a squaregrid pattern on 0.91-m centers. Before placing the cngeon the green surface,thirty entry holes were made in the flrrfunder each iug" by i"t"tti"g nails into the turf. Fifteen larvae were placed on the turfgrass surface within eachcagJat 09:00 h and gently sprayedwith tap water beforethe cageswere placed over the larvae. To measurethe effects of a shade gradient that existed from the tl parvifolia trees,four treatments,(Notth OutsideRow = NO, North Inside Row = NI, South Inside Row = SI and South OutsideRow = SO) were aligrredperpendicular to the gradient and replicatedeight tirnes in an eastwest direction parallel to the gradient. Cutworm establishmentwas evaluatedsix and 24 hours after infestation to estimate establishmentand recoverability of the lan'ae. The turf surface rmder each cage was drenchedwith a disclosing solution (l0ml Joy Ulfra/7.57 liten of water) and all larvaethat carneto the surfacewithin 5 minuteswere couoted. The numberof lanral cadaverspresent on the turfgrasssurface was also recorded. Treatmentmeans were separatedusing PROC ANOVA (SAS Institute, 1985)and Fisher's ProtectedLeast Sigrrificant Test (P 10.05). Experiment2. This experimentwas initiated in responseto the ant activity obsened in Experiment l. It was designedto progressivelyreduce access into or out ofthe cagesby ants and other artbropods. Plos were establishedon the turf in a squaregrid pattem on 0.91-m cent€ts. An estimateof ant mound density was tak€n by counting ant nestsin the study areapriot to cageplacement, so that we could avoid placing a cageon a visible ant mormd. Fifteen secondand third instar black cutwomr larvae were confined to eachplot using cagesand methodsdescribed previously. The cagesremained in the sameplace for the duration ofthe experiment. This 2x3 fastorial experiment was conductedtwice, on 20 July and 20 August at different sites on the study green. It was ananged in a split-plot design with four replications. The main factor was time of infestation and the sub-factorwas type of cage barrier. Infestations occurred either at 12:00 h (daytime) or 2l:00 h (nighttime). Exclusion teatnents consistedof: l) cagesplaced directly on the turf sruface;2) cages placed into a ring of foarn stravingcream (Gilletter9; and 3) cagesplaced into a ring of shat'iog cream that also encompassedan inner plastic barrier (4-cm in height x 25.5-cm diameter x l-mnr thick) which was vertically buried 1.5-cm deep into the soil srrface. Additional shavingcrcarn was usedto fill the spacebetween the irmer barrier and the cage. Shaving cream was used becauseit provides a temporary barrier for containing larvae within, or excluding surface-crawlingarthropods from enteringthe study area,yet doesnot injure the bentgrass(Weinhold et al. 1998). The number of surviving cutwonns was evaluated36-38 hours after infestation by drenchingeach cagearea with a disclosing solution (20-ml Joy Ulfra/7.57 liters of water) and counting all live larvaethat cameto the surfacewithin 5 minutes. Dead larvaebodies were also counted. Any visible ants found on the lanrae cadavemwere collected for identification. Analysis of teafinent effects was determinedusing PROC MDGD (SAS Institute, 1985) wlrere the month that the study uras conductedwas treated as a ralrdom effect Treatn€Nrtm€ans were comparedusing Fisher's Protectedleast Sipificant Test (P < 0.05). 33 RESI.JLTS Experiment1. The six-hour post releaseevaluation is reportedbecause no larvae were found in the 24-h sample. There was a sigrificant (Fl5.6l; df1,3; F0.0001) directional pattern of lanal survival that we athihrte to the presenceof sHe. The northemmostset of cages,designated NO, containedan averageof 4.75+1,17live lawae per cage(Table l). The next adjacent row of cages, designatedNI, contained an av€rageof 0.6t0.67 live lanraeper cage. The SI and SO set ofcages wlrere shadewas absentdid not contain any living larvae. There were significant (F =14.48; dF-3,3; P 4.0001) differencesanrong treatm€ntsfor dead larvae. In this casethe southernmostset ofcages (SO) containedno cadavers. The next three sets containeddead larva bodies, which" when examined,were neady always entirely coveredby ants. ln somecas€s, the antshad to be dislodgedto find the larval cadaver. Someofthe live larvaewere also being attackd by antC and exhibited repeated thrashing motions which appearedto be in responseto the ants. All ants associatedwith living or dead larvae were collected and later identified as S. malesta. lt appearedthat predationactivity of S. molestawasinfluenced by shadeintensity; visible ant activity was mone noticeable on dead larvae that were in firll sun compared to dead cadaversthat were in full shade. TABLE 1. Number of A. ipsilon larvaerecovered six hours after releasein cagesarranged to evaluateeffects ofshade on larval survival and recoverv. Shade Level CagePlacement LiveLanaelcageu Deadlarvae/cageu Mean Ct SE) Mean (!SE) Shaded North Outer 4.75(r.r7) a 2.88(0.86) b North Inner 0.63(0.67) b 7.63(r.r4) a tI SouthInner 0.00(0.00) b 5.38(0.92) ab Sunny SouthOuter 0.00(0.00) b 0.00(0.00) c oMeans within a column followed by the sameletter are not differ€nt accordingto Fisher's Prcr€credLSD (P > 0.05). Experiment 2. Frequent and low mowing disrupted the featlues of ant mounds; however,a pre-experimentalevaluation of the numberof ant mormdspresent indicated that ant colony density was ca.2 pet nf. Not all deadlarvae had antsactively feedins on thern, but when prcs€nt,all ants were identified as S. molesta. Occasionally,dead lanae were fomd along the shaving ctean border, and apparentlydied fiom their physical encounter with the shavingcreanr. There was no significant difference in lanral survival all a r€sult of the time of release (F:3.41, dFl,28; ^F0.ll) but ttrere was a differencein larval survival that could be attributed to the tlpe of exclusion system used (F172; dts228; F0.0001) (Table 2). There was no significant interactionbetrveen cage and time of release(F = 1.07, df : 2,28, P=03a\. There was no significant difference in the numberof recovereddead larvae attributable to the time of release(Fr2,.51; dts1,28; F0.16); however,there was a difference in the number of recovereddead larvae that could be attributed to the tne of exclusion system used .Ft8.29; d?1,28;.F0.003). There was a significant interaction betweencage and time of release(F=1.07; dF2,28; F0.34) with rcgard to dead larvae recovered. Results showedthat more dead larvae were recoveredin the cageswith shavingcream than were 34 reeoveredin tbe cage with shaving cream and a plastic barrier when releasedat night, urhile that hend was not evident in the cageswith lawae that were releasedin the day. When live and dead larvae werreadded togeth€r, there was a sigrificant difference in the total number of recoveredlarvae dtributable to the time of release(F=8,A; dFl,28; H.A). There was a difference in the total number of rccov€r€d lawae tha could be atuibuted to the type of exclusion system used (F =17.91; dts2,28; F0.003), and a significant interaction betweentime of releaseand cageWe (F=7.16; dts2,28;.F0.003)' Again, results showedthat more total lanrae (alive + dead) were recoveredin cageswith shaving crean if the larvae were releasedat night than were recovercd in cageswith shavingcream and a plastic barrier. We cannot explain that interaction,but we believe it to be an artifact ofthe placementofthe cagesover ant coloniesor egresspoints that were not visible when the cageswere placed. TABLE 2. Influence of infestation time and €xclusion method on srnival of black cutworm larvae 36 hours afterlelease. Live lawae/cagec Deadlarvae,/cage" Totallar,tdcagec Treatnenta'o Mean+ (SE) Mean+(SE) Meant(SE) Day C 0.75(0.63) b 0.63(0.53) b 1.38(0.8a) c DayC+S 1.25(0.85) b 2.38(r.44)b 3.63(1.01) bc Day C+S+B 6.25(r.64) a 2.s0(r.23)b 8.75(0.47) a NightC r.00(0.64) b 1.25(0.63) b 2.25(l.M) c Nightc+S 4.50(1.53) a 7.80(1.21) a 11.88(1.12) a Nightc+s+B 7.50(3.35) a 0.90(1.69) b 8.38(0.45) ab Day (Combined) 2.75(0.78) ns 1.83(0.63) ns 4.58(o.79) a Night (Combined) 4.33(0.83) ns 3.17(0.85) ns 7.50(0.69) b C (Combined) 0.88(O.af a 0.94(0.43) a 1.82(0.31) a C+S (Combined) 2.88(0.83) a 4.88(1.26) b 7.76(0.76)b C+S+B Combined) 6.88(1.08) b 1.69(0.68) ab 8.57(0.76) b " Infestntionsoccuned either at 12:00 h (day) or 2l:00 h (night) on the 20 July and20 August 2002. D C = plastic cage, C+S = plastic cage+ shaving cr€am,C+S+B = plastic cage+ shaving cl€an + inner plastic buried banier " Datare meansof eight replications.Means within a column followed by the sane letter are not different accordingto Fisher's ProtectedLSD (P > 0.05). DISCUSSION Solenopsismolesta is a widely disributed inhabitant of pastureand Fairie landscapes in the US and is also a cornmon ant of urban and turfgass landscapes(Cocldeld and Potter 1984, Zengeraod Gibb 2@l). Becauseof its srnall size and subterraneonnatur€, S. molestais rarely noticed exceptin areassuch as short-mowedgrass landscapes, where it is readily observedon the soil surface. Early studiesindicated M. S. molestafed prinrily on seedsand occasionallyinseots (tlayes 190); however, more recently S. molesto was rcpoted to be a commonpredator of eggsof Japanesebeetle and southetamasked chafer (Zengerand Gibb 2001) and black cutworm (Lopez and Pott€r 1999). 35 Solenopsismolestawas the only ant speciesobserved feeding on cutworm larvaein the study areaand appearedto be an important factor affecting sunival of the cutworm larva€. The type of confinementcage used in this study was similar to thoseused to confine black cutwonns in other insesticidescreening studies. In those studies,the cageswere intended to protect the cutworms ftonrprtdation by birds (Heller and Walker 2002). T\e cagesare also suffrcient to prevent the small, first-instar cutwonn larvae fiom €scapingthe study atea. Due to their size and zubterraneannature, S. molestanumh could not be accurarcly determinedvisually within the cag€s. However, differencesin ant numbersand activity were easily apparentamong cages. Dead larvaewere oftsn completelycover€d by ants in somecages, whereas other larvaeremained alive and were attemptingto dislodgeattacking ants by thrashing their body on the turf surface. Lopez and Potter (2000) also observed black cutworm larvae exhibit this defensivethrashing behavior in responseto attacksfrom larger ants,such as ZasiasneonigerEmery. The cagesdid not effectively deter ant movern€ntinto the cage interior without the installationof additionalbariers. The addition of shaving cream bet\ileen the bottom of the oageand the turfgrasscanopy was employedto preventS. molestafrom gaining access to the interior of the cage through surface movement. The cage and shaving cream collapsed the turf canopy and bonded individual leaf blades together. In addition, the shavingcr€am rnay also have possessedchemicals urhich preventedot maskedthe ability of the ants to find larvae, which may partly explain why more cutwonns sunrived in the cagestreated with the additional shaving cream barrier. However, S. molestawere still observedinside someof the cages. Somecutworm larvaedied after contactingthe shaving creasrbarier. Theselarvae were not observedto be preyedupon by ants. To prevent S. molesta fiom gaining access to the cages thtough subterranean movement,a plastic inner barrier was forced vertically 1.5-cmdeep into the soil. Shaving cream was used as an additional barrier to prevent any artbropod galning accessby crawling over the plastic banier. Despite these efforts, black cutworm lanae (alive or dead)were physically absentin three cages(one in July and two in August). The nestsof this specieswere often difficult to detect. According to McColloch and Hayes (1916), S. molestanests are difficult to locate becauseof their sma[ openingswhich frequenfly ale located somedistance from the tnre nest. They also reportedthil S. molestanests may be connectedto nest ofother speciesby long undergrotmdgalleries. The absenceofcutworm larvae with no other visible evidence of disnrbance to the turf surface can only be attributedto predationby an arthropodpredator which bad unrestrictedsubteranean access to the interior of the cages. The only predatorwe saw feeding on lantae was S. molesta. During Experiment l, we observedseveral cadavers,which were covered by S molesta, disappearwithtn 12-24 hours, as the ants canied the r€Nnnantsof the larval cadaveraway. The 36-h intErval that occurred from release to fust data collection in the second experimentwould haveprovided ampletime fot S. molestato rsmovecaptured lalvae. Weatherconditions during thesestudies are reporrcdin Table 3. Infestationsoccurred either at 12:00 h (day) or 2l:00 h (night) on 20 July eq:l 20 August. Ternperatureswere quite higb during the experiments,ranging fiom 74-100" C. It is plausiblethat survival of tlre cutworms was lessened by these high temperatures.How€ver, lhere were no differences in sundval attributable to time of release. Weather conditions do not effectively explain the increasedsurvival of cutwormsthat occurredas the banier became mor€ r€strictive, nor do they explain tbe increasedItt€s€nce of intact dead cadavers, presumablynot removed by S. molesta, tbat were found in cageswith more rpstictive baniers. The most plausible explanationwas that the barriersresticted S. molesta apcess to the larvae resulting in more sunriving lanae, and more total larvae (live + dead)being recover€d. 36 TABLE 3. Weafherconditionsa during Experiment2. 20 Jul 96 74 82.7 0 2l Jul 96 74 82.8 0 22 Jul 100 75 82-9 0 20 Aug 96 74 82.s 0 2l Ang 95 80 81.9 0 22 Atg 98 77 82.5 0 I Data from the OklahomaMesonet@, http://agrrreather.mesonet.ors b soil temperaturesr€corded to- *a*@. Ants ale often regaded as a nuisancepest on golf coursesdue to the lack of awareness ofthe potential benefit they provide in suppressingpopulations ofdarnaging insect pests. As a result, ants ar€ either targeteddfuectly or not consideredufien pesticidesare chosento conhol other pestsin the furfgrasslandscape. A noticeablereduction in ant predationwas observedby Lolez-ad Potter (2000) following the useof a broad spectruminsecticideand indicated that ants provide an important futff61 against pest outbreaks in the turfgrass environment. This study iudicates that S. molestamay be a potentially important predator of black cutworm larrraeon golf corrrsegr€€ns. In this seriesof experiments,cutworm larvaewene introducedonto the bentgrassgreen at levels that would causeconsiderable visible damage to the turf, yet they were virhrally eliminated from the greenwithin 24 hours, presumably through predationby s. molesta. This certainly suggeststhat predationby s. molestalns the potential to reducethe risk ofblack cutworm infestationsas well as other turfoests that feed on golf coursegreeDs. The colony density of S. molestain this study 1Zlm2;-ishigh€r than densities reported in native prairie locations (caangui et al. 1996), and the high density may explain why p,redationof cr$vvomrlarvae seemedto be so prevalenton our shrdy site. Judiciousselection and use ofinsecticides may €ncouragethe establishnentofants in selectedareas of the trrfgrass environmentand enhancetheir beneficial activities A,oWz and Potter 2000). Howw€r, the use of broad-spectruminsecticides in many intensely managed trufgrasses may be responsible for reducing ant populations and therefore perrtitting pest establislrment(Terry et al. 1993). Further study is neededto determine potential benefits and limitations of ant presencein highly managedturf, and how they misht be effectively into a complehensivepest rumagement system for hrrf managementin golf courses. ACKNOWLGEMENT This manuscriptis publishedwith the approvalof the Director, OklahomaAgriculoral. Experiment Station, Stillwater. The project was partially supportedby the Oklatroma Agricultual Experiment Station Project 2420 and the Oklahoma Turfgrass Researph Foundation. Thanksto J.V. Edelsonand K. L. Giles for reviewing an eadier draft of this manuscrip. JI LITERATI.JRECITED Anonymous. 1999. The aulity of Our Nation's Waters,Nutients and Pesticides.U. S. Geol. Surv. Clrc. 1225. Baxter, F. P., and F. D. Hole. 1967. Aat (Fotmica cinerea) pedoturbationin a prairie soil. Soil Sci. Soc.An. Prcc.3l:425428. Canrpbell,G. 8., R l.Brazne,A. G., E. T. B. Voigt, D. F. Wamoch and J. L. Hall. 2001. The Illinois Green Industry: economic impact, structule, characteristics.Univ. Ill. NRES Report.200l-01. Cantangui,M. A., Fuller, B. W., Walz, A. W., Boetel,M,A., and Brinkam,M. A., 1996. Abtmdance,diversity, and spatial distribution of ants (Hymenoptera:Formicida) on mixed-grassrangelands treated with diflubenzuron.Environ. Entomol. 25:757-76. Cocldeld, S. D., and D. A. Potter 1983. Short4ermeffects of insectioidalapplications on prtidaceous artbropods and orbatid mites in Kentucky bluegrass ttrf. Environ. Entomol. 12:1260-126/'. Cockfield, S.D., and D.A. Potter. 1985. hedatory in high-rd low- maintenanceturfgrass. Can. Entomol. ll7 :423429. Hayes,W.P. 1920. Solenopsismolesta Say (Hym.): A biological study. Kans.Agric. Exp. Sta.Tech. Bull. 7:1-55. Heller, P.R, and R. Walker. 20A. Suppressionof black cutwom larvaewith Talstr and Tempo formulations on creeping bentgrass,2001. Artll Mgt. Tests G3: Elechonic Publicationhttp ://www.entsoc.ore/. Kunkel, B.A., D.W. Hel4 and D.A. Potter. 1999. Impact of halofenozide,imidacloprid, and bendiocarbon beneficial invertebratesand predaroryactivity in turfgass. J. Econ. Entomol. 92:922-93O. Lopez, R., and D. A. Potter.2000. Ant predationon eggsand larvaeof the black cutrvomr (Lepidoptera:Noctuidae) and Japanesebeetle (Coleoptera:Scarabaeidae) in turfgass. Environ. Entomol. 29:116-125. McCollucb, J.W., and W. P. Hayes. 1916. A preliminary report on the life economyof Solercpsis molestaSay. J. Econ. Entomol. 9:23-38. Potter, D.A. 1998. Destnrctive turfgrass insects. Biology, diagnosisand control. Ann Arbor Press. Chelsea,MI. Terry, L.A., D.A. Potter, and P.G. Spicer. 1993. lnsecticidesaffect predatoryrthrcpods and predationon Japaneseb€etle (ColeopGra:Scarabaeidae) eggs and fall m)'worm (Lepidoptera:Noctuidae) pupae in orfgrass. J. Econ. Entomol. 86:871-878. SAS krstitute. 1985. SAS usersguide: statistics,version 5 ed. SAS Institute, Cary N.C' Wang, D., K. Mc Sweeney,B. Lowery, and J.M. Norman. 1995. Nest strucfiueof the ant Lasiusneoniger Emery and its implications to soil modification. Geoderma66:259- 272. Weinhold, A.P., F. P. Baxendale,and R D. Grisso. 1998. Mechanical control of late instar black cutworm on bentgrassusing a John Deere RZI700 high pressureliquid injection applicator. Arth. Mgtt. Tests24:340. Z,etger, J.T., and T.J. Gibb. 2OOl. Identification and impact of egg predators of Cyclocephalalurida nd Popillia japonica (Coleoptera: Scarabaeidae)in turfgrass. Environ. Entomol. 30:425430. 38 vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 SEASONALBIOTOGY AND ASSOCIATED NATURAL ENEMIES OF TWO TOUMEYEIf,./I SPP.I IN COLORADO Dayna D. Cooper2 and Whifirey S. Cranshaw3 Departmentof Bioagricultural Sciencesand Pest Management Colorado State University, Ft. Collins, CO 80523 ABSTRACT Observationswere made during 1994 and 1995 on the life history and associated natural enemies of two Toumeyella spp. of soft scale which recently have become establishedas important pestsin severalColorado communities. Observedhost plants of the stri@ pine scale, Towneyellapini (King), included pints sylvestrk, pinus mugo, Pinus edulk and Piruu nigra; obsr;rvedhosts of the pine tortoise sale, Toumeyella parvicornis (cockerell), were Pinus contofia and p. sylvestris. Both species were gbryrved to have only a single generationper year. crawler emergenceof r. pini bqan in Denver on I June n 1994and 21 Junein 1995. This was close to that observedwith T. parvicornis in loveland and Greeley, 27 lo.dayin 1994and 2l June in 1995. crawlers were present for about one month. No parasitoids were recovered from z. pini, bnt pr€datorsof early instar stagesincluded the coccinellidsHippodonia convergens(Guerin- Meneville), coccinella EeptempunctataL. and a predatory lampyrid, Lucidota sp. conversely, an Apltytis sp. aphelinid wasp may be important on populations of p. pamicornis. INTRODUCTION Two speciesof pine-infesting soft scalesof the genus Towneyellahave colonized many easterncolorado communitiessince about the mid-1980's. Both roumeyellapini (King), the striped pine scale, and roumeyellaparvicornis (cockerell), the pine toriise scale, are widely distributed in e.Ntern North America where they are recognized as important pests of forest trees, shade trees, christmas tree planiings and pine seed orchards(Clark et al. 1989,Sheffer and Williams 1990). Although not previously recorded from the Rocky Mountain region, both have emergedas significant pestssince their establishment. Most severehas been the effect of T, pini, particularly on Pinw sylvestris, which has severely damagedand even killed plantings over the past decadein the Denver area and in severalother metrooolitan areas in easternColorado. In addition, theseinsects produce abundantamounts of troneydew that attract nuisancewasps and beesand seriously degradeplant appearancedue to growth - Homoptera: Coccidae 'I Currently at 13555N. Sandra - Rd., Marana,AZ 85653. Department of Bioagricultural Sciences and pest Management, colorado state University, Ft. Collins, CO 80523. 39 of sooty mold fungi. The severity of this scale injury has produced a need to better undentand the regional biology of these introduced pest speciesas a meansto develop more effective managementapproaches. Relatedto this, purposesof this study were to determinethe numberofgenerations annuallyproduced, identify critical periods in the life history (e.g., crawler stages)and documentthe incidenceof biologicalcontrols. METHODS AND MATERIALS Berause of taxonomic confusion about the Toumeyella present in the state (Cranshawet al. 1993) positive speciesidentification was an initial effort. Both T. pini and T. panicornis viere identified to speciesusing previously described dorsal habitus characteristicsof mature male exuviae (Williams and Kowtarab 1972). Life History Smdies. Life history studieswere conductedat four sites, two infested with 7. pini and the others infested with ?. parvicornis. Survey sites used during this study were: a municipal park (Orchard Park) located in central Denver, Colorado, with heavy infestations of T. pini on P. sylvestris; two adjacent southern Denver parks @osamundPark, Southmoor Park) with T. pini on P. nigra Oghtly infested) and P. sylvestris(heavily infested); a Loveland, Colorado, residencesupporting populations of ?. partticomis on Pinus nigra (havy infestation) and P. sylvesrris (moderate !o heavy infesations); and a Greeley, Colorado, botanic collection (Housten Gardens) with moderateto heavy infestation of T. pamicomis on Piruu contortct. Site visits were initiated 17 March 1994 and concluded27 September1995. In both years, sampling was intensified to three !o four times per month during the active crawler period, I June through 30 June L994 and I Junethrough 2 August 1995. For determinationof crawler activity tap sampleswere madeby striking one branch terminal, at each cardinal point, four times over a 33-cm x 40.6-cm white plastic tray. During visits to these sites, casual observation of the most consistent and obvious flowering herbaceousand woody perennialswas madein an effort to locate plants with phenological similarities ta Toutneyellaspp. scales. Natural EnenrySumeys. Evaluationsofpredators were madeduring the courseof the tap sampling and were supplementedby visually observing speciespreying on scales. Additionally, a checkof parasitismwas madeusing a seriesof I0 ta 12 infestedterminals collected during each sample, beginning 1 June 1994. All other insect specieswere removed from the terminal and a 4-cm section of infested curent seasongrowth was placed in a cotton stopperedvial and maintainedfor parasitoid emergence' RESULTSAND DISCUSSION Life Hktory: Towneyellapini. Observationsmade from Georgia of striped pine scaledocumented multiple generations(Clarke et al. 1989). Three overlappinggenerations were suspectedwith peak crawler abundanceoccurring May to early June, mid-July to early August, and Octoberto early November. In Coloradoduring the courseof this study all T. pini colonies producedonly one generationper yeal. ln 1994, T. pini crawlets were first detectedI June. Eleven days prior to freld emergence(22May) eggs examinedin the laboratory had visible eye sPots,an indication observed to indicate that eclosion was imminent. At time of crawler emergence'new needlesof the Pinus spp. host plants had not fully elongated. .Crawlerswere observed settling on immature ne€dles,cones, and twigs. Small numbersof crawlers were still being observed21 June,but all crawler activity had ceasedby l1 July. ln 1994, emergenceof adult male striped pine scales, which were restricted to 40 still needles,was not observeduntil samples tilken 2 August. At this time, most males "pupal occupied- their cocoonst; all had emergedby mid-Seprember' In 1995,-crawlerswere not detectedin ttre ReU until 23 lune,22 days later than the 1 Juneemergence of 1994 (Fig. 1). This postponedemergence is suspectedto result the spring of from unseasona6ly-ofcool, wet wea-theiconditions ihat persisted throughout iSSS. thir type dehy in activity by crawlers has been reported to occur with other speciesof Toirneyeltaas i result of adverseweather conditions and microclimate (Wallner t'szg). Low nurnb"tr of active crawlers were still being obsenredin early July with all co*iet activity ending subsequentto the 17 July sample' The duration of the emergence period (approiit"t"ty-fO days) was similar to the observedemergence period of 1994' 35 a3 I .ad e J l X I 9,,q € I a21-*u; i i ffi siter I 3'16B L 1.5 -J K ll, ' gl U 1r 0.s at) 0 2l Jun FIG. 1. T. pini (strr@ pine scale)crawler emergencepatterns from sites C and D, 2l June to 25 luly 1995. Values on the y axis are a rating scale measuringnumber of crawlers dislodgedduring tap samples(0 : no crawlers, 1 : 1-50 crawlers, 2 = 51-100. crawlers, 3 = 101-500crawlers, 4 = 500* crawlers). Site A = Orchard Park, Denver, Colorado; Site B : RosamundPark and SouthmoorPark, Denver, Colorado. During both years of this survey, Centranthus ruber L. (Red Valerian) was consistently in full bloom at the time of crawler emergence,This observation appears significant when consideringthe number of days that se,paratedthe two emergencedates.- Tierefore, Red Valerian may be useful as a phenologicalindicator to predict the onsetof crawler emergencefot T. Pini. Based on personal observationsmade during the course of this study and from reports of area landscapecare professionals,P. sylvestriswas the most common host of T. pini found in northeastemcolorado. However, P. nigra, P, edulis and P. mugo e'an also support T. pini infestattons. At somesites severeinfestations were observedto result in rapid decline of susceptiblehost plants within two years. 41 Lrfe History: - Tornneyellaparvicornis. Although T. parvicornis hasbeen recorded to have either one or two.generations/season (wilson rszrl, only " ,ingi"l"*otion was observedduring this study. During the initial survey (17 March 1gg4), no eggs were presentbeneath the body of the female. crawler emergencewas fust o6served27 May at both sites. Emergence reachedits peak during the irrst week or rune-ano continued through the middle of the month, with a very sriall number of crawlers still active 23 June' Examination of overwintered femalesit this tir" indicated no uiauie eggs ar one and a very few live $te eggs and newly hatchedcrawlers beneaththe female exuviae at the second gv yJ9. 30 June, no crawlerl could be found at either site. observationsmade 17 August 1994at both sitesrevealed instar III femalespacked .fong il e irigs of infested branches. In . . ^1f95, samplings made I and 14 June revealed eggs prcsent within the body cavities of females, but no nymphs were present under thJfemhes. crawlers activity \gn^2L June, approximately 25 days later than the recorded emergence date for L994 (Fig. 2). Numbers of scale crawlers dropped by early July, but "o-*to, were detected througfr 25 July at both sites. a0 € fr (l} t f; 6t qg L iu 3 6l tr U Ir qt u'5-' (aTJ 0l-,,,,-.,,,-- l4 Jun 21 Jun 3 Jul 18 Jul 25 Jul Dat* IIG. 2. T. parvicornis @inetortoise scale)crawler emergencepatterns from sitesA and B, 14 Juneto 2 August, 1995. values on the y axis are a rating-scalemeasuring number of crawlers dislodged during tap samples(0 = no crawlers, 1 = l-50 crawlers, 2 = 5l- 100 crawlers, 3 = l0l-500 crawlers, 4 = 500* crawlers). site c = toveland, Colorado;Site D : HoustenGardens, Greeley, Colorado. Emergencehas been reported to continue for approximately 13 days (Rabkin and Irjeyle 1954), beginning in mid to late June. During 1994, crawier emergencebegan in late May and persistedfor 35 days, althoughnumbers decreased markedly during the tast 42 week. In the subsequentyear, marked by an unusually cool, wet spring, a mid-June crawler emergence*as noied, although crawler activity again extendedover a month' Tap simples from 2 August showedno crawlers presentat either site. Subsequent observations made27 Septemberfound that male emergencehad occurred. Vacant male tot. *"t" found packedin amongfemale scalesalong current season'sgrgwth. Very few males were ever obsenyedto setile on needles. No evidenceof a secondgeneration was ever Observed. Naural Enenry obsentations: Towneyellapini. The most commonly observed - insect predators of f. pini populations were beetles. Two species of coccinellids Hippodarnia convergens(Guerin-Meneville), the convergentlady beetle, md Coccirwlla teptempurrctataL., tlie C-7 lady beetle - were observedfeeding on nymphs in lale June oi tgg+ and early-beetle, July of 1995. Another qpeciesofcoccinellid, Adaliabipunctaa L., the twospottedlady was also presenton T. pini infested trees but was not specifically observedto feed onT. Pini. A lampyrid predator was also commonly observedduring the sametime period as the lady beetlei. Members of the Lampyridae are not known to be important predalors of immature scale insects and many iue not reported to feed in the adult stage (Arnett 1960). Howwer, Lucidnta sp. adults were observedwalking over needle surfacesand feeding on immature stagesof T. pini duing both years of this survey. Predationby birds may also be important as natural controls of striped pine scale. The pine warbler, Dendroica pinrer (Wilson), has beenobserved feeding on stiped pine scaleinfested Scotspine in Boulder, Colorado, in 1992and 1993 @eldstein1993). Othet avian predators of T. pini that have been observedinclude the yellow-rumped warbler, Dendroica cororuta (L.), and the house frnch, Carpodacu'srnexicaruts (Muller). These avian predators focus their searcharound needlebases, which is the area most intensely populatedby both stripedpine andpine tortoise scalefemales @ave I-eatherman,Colorado State Forest Service, personalcommunication). A number of parasitoid specieshave been reported to account for up to 15% parasitism in Georgia T. pini populations(Clarke et al. 1989). However, no parasitoid wasps were recovered from laboratory vial samplesduring the course of this survey. Clarke et al. (1989) reported that parasitoidsappear to have little effect on controlling T. pizi populations even when they are abundantas parasitizedfemales remain capableof producing large numbersof offspring before parasitoidsare fully developed. The most important natural enemiesof T. pini that are reported to occur in the southernUnited Stateswere not found in Colorado. A pyralid larva, Inctilia coccidivora (Comstock), is reported as a particularly efficient predator of female striped pine scales in Georgia (Clarke et al. 1989). This speciesoccurs throughoutthe eastern,southern and southwesternUnited Statesbut has not been reported from within the Great Plains region (Heinrich 1956). Narural Enemy Observations: Towneyellapamicornis. Six qpeciesof lady beetles are reported as important predators of pine tortoise scale in Ohio and Mnnesota. Of rhese,Hyperaspis congressis (Watson) (: biorcta Say)is reportedto be the most abundant and important (Orr and Hall 1931, Wilson 1971). This speciesof Hypetaspis is not known to occur in Colorado and during the courseof this survey only one speciesof lady beetle, EL convergens(the convergentlady beetle), was observedwithin pine tortoise scale colonies. The importancn of H, convergew as a natural predator of T' pawicornis remains unknown, as adult beefleswere only seenoccasionally and at only a single site. Evidence of parasitism was found within colonies of pine tortoise scale at both sites. A total of 52 vials of scalesused for rearing parasiioidsduring the course of one season,27 May through 23 June, yielded nine adult parasitoid wasps. These parasitoids 43 were identified as an Aprrytis p. (Hvqeloptera: Aphelinidae) (Boris Kondratieff, Department.of Bioagriculturar Sciencesina pot Managiment, cio.aao-iate, personal communication). Ttris is a common and abundant ginus oi soft and armored scale pTTi9]91: although appargntrynot previously r@rt"d from z. pawicornis (Krombein et al' 1979). voucher specimenswere placed-in the c.p. Gillette Museum of Arthropod Diversity at Colorado Sate University, Fort Collins, Colorado. Early reports list a single parasitoid species,Microterys fuscicornis (Howard), as being present in Minnesota pine tortoise-scale populations lorr ano rru teitl. \lcrgteUs fusicornis, along with the parasitoids Gotnniella iaisoettiae Timberlake, Chciloncurus sp', and Tetrastichussp. were later reported to attack both ?. parvicornis and pini r. when mixed infestationsoccur (Clarkeet al. 19g9, wilson l97lj. None of thesespecies were observedin this study. rn p. . summary, the life cycle of both pini and p, parvicornis in colorado is univoltine, unlike that reported in someother locations. Intiition of crawler activity for both speciesoccurred at approximately the sametime but crawler activity for bottr,was three weekslater in the secondstudy year, emphasizingthe importanceof weatheron this aspect .of derrelopment. crawler activity also was extended, typically persisting for approximately one month. Parasitoidsappear to be significant natutA-cont ols for Z. parvicomis but not for T. pini. predations by coleoptera and birds were the most important naturally presentbiological controls of the latter. Thesedata have subsequenfly been incorporaled into Extension publications for the Rocky Mounain stat€s(Cranshaw et al. 2000). ACKNOWLEDGMENT We would like to acknowledgethe assistanceof Drs. Boris Kondratieff and David R. Smith for their assistancein identification of the insect specimensdiscovered in this study' Funding for this project was assistedby Colorado Agricultural Experiment Station Project618. LITERATURE CITED Arnett, R.H., Jr. 1960.The Beetlesof the united States. catholic univ. of Amer. press, WashingtonD.C. pp. 547-549. Clarke, S.R., Debarr, G.L., and C.W. Berisford. 1989. The life history of Towneyella-Can. pini (Kng) (Homoptera:Coccidae) in loblolly pine seedorchards in Georgia. Entomol.121:853-860. cranshaw,w., D. katherman, andB. Kondratieff. 1993. Insectsthat Feedon colorado Treesand Shrubs. ColoradoState Univ. Coop.Ext. Bull. 5064. 197 pp. cranshaw,w., D. I-eatherman,L. Mannix, andw. Jacobi. 2000. Insectsand biseases of Woody Plantsof the CentralRockies. ColoradoState Univ. Coop. Ext. Bull. 506A. 283 pp. Feldstein,s. 1993. Pineand palm warblersin Boulder duing r9921L993winter. c.F.o. Journal 27:137-139. Heinrich, c. 1956. Americanmoths of the subfamilyPhycitinae. u.S. Nat. Mus. Bull. 207. SmithsonianInst,, Washington,D.C. pp. 230-235. Krombein,K.V., P.D. Hurd, Jr., D.R. Smith,and B.D. Burks. 1979. Catalogof Hy menopterain North America North of Mexico. Vol. 1 Symphyta and Apocrita (Parasitica). SmithsonianUniversity Press., Washington, D. C. Orr, L.W., and R.C. Hall. 1931. An experimentin direct biotic control of a scaleinsect on pine. J. Econ. Entomol. 24:1087-1089. Rabkin, F.B., and R.R. Irjeune. 1954. Someaspects of the biology and dispersalof the pine tortoise scale, Towneyellaru'ttnivnaticwn @ettit and McDaniO (Homoptera: Coccidae).Can. Entomol, 570-574. Sheffer, B.J., and M.L. Williams. 1990. Descriptions,distribution, and host-plant records of eight first instars in the genus Tbwneyella (Homoptera: Coccidae). Proc. Entomol. Soc. Wash. 92:44-57. Wallner, W.E. 1978. Scale insecs: What the a$oriculturist needsto know about them. J. Arboric. 4:97-103. Wilson, L.F. 1971. Pine tortoisescale. USDA ForestPest IJaflet 57. 7 pp. Williams, M.L., and M. Kosztarab,1972, Insectsof Virginia no. 5; Morphology and systematicsof the Coccidae of Virginia, with notes on their biology. Virginia Polytechniclnst.St. Univ. Res.Div. Bull.52.,2L5pp. 45 vol,.29 NO.1 SOUTHWESTERNENTOMOLOGIST MAR.2004 NOVEL BEAUWNA BASSIANADELIVERY SYSTEM FOR BIOLOGICAL CONTROL OF THE RED IMPORTED FIRE ANT' Blake R Bextine, and Hartan G. Thorvilson College of Agricultural Sciencesand Natural Resowces DepartmentofPlant and Soil Science TexasTech Univenity, Lubbock, Texas79409 ABSTRACT The red imported lire ant (solenopsis iwictaBtren;Hymenoptera:Formicidae) is a major pest throughoutthe southemUnited States,in part, becausefew natural enemiesare present. The entomopathogenicfungus, Beaweria bassiana,caused significant mortality in the laboratory,but the funguswas less effective in field trials, perhaps becauseants did not remain in contact with fungus long enough to become infected. In previous e4periments,S. invicta removedunbaited fungal alginate pellets that were placeddirectly into rnounds,resulting in no reduction in fre ant populations. However, baiting pellets with peanut oil improved delivery of the biological control agent and reduced fire ant populations in heavily infested cattle pastures. In the study reported herei4 we have developedanother method of B. bassianadelivery directly into fire ant nrounds,targeting individual colonies. Placing woodencraft sticks or woodenmarking stakescoated with 8. bqssianaeffectively killed fire antsand renderedmost moundsinactive. INTRODUCTION Beannteriabassiona (Balsamo) vuillemin, an entornopathogenicfungus, shows promise as a biological control of red imported fire ants, Solenopsis iwicta Btfierl (Hymenoptera:Formicidae), by causing mortality in all stagesof development(Broome 1974;Stimac atal.1990,1993; oi etaL l9g4). The introduction of .8. bassiana rnto S. invicta laboratory colonies maintainedin soil caused significantly less mortality than in colonies without soil. However, encapsulationof mycelia in alginate pellets allowed the fungus to suryive better in soil @ereiraet aL l993Lb: white 1995). Ants did not abandonsand that was contaminated with A' bassianaconidia in laboratory studies,increased mortality was not observed,and dead aduhs were removed from colonies before fungal conidiogenesis. B. bassiou was recovered from fourth stage larvae that had been fed buccal contents bv aduh ants (Seibeneicheret aL 1992). I Solenopsisinvicta Bven;Hymenoptera: Formicidae lDepartmentof Entornology,Univenity of Californi4 Riverside,CA 92521 4' 7 In field trials, introduction of unbaitedalginate fungal pellets did not significantly reduce s. invicta populations,and fire ants removedpellets from mounds@exine 1998, Bextine and Thorvilson 2002ab). when coated with peanut oit however, pellets were retainedin mounds,and meancolony ratings were significantly reducedwithin two weels. Successfuldelivery of B. bassianata S. invicta colonies over extendedperiods of time is necessaryto causesignificant mortality and reducepopulations. Our objective was to determineif placementof immovablewooden sticks and stakescoated withB. bassiana into S invicta moundswould causemortality of colonies. Beoauseants would be unableto removethe sowce of inoculurq exposureto B. bassianaconidia over long periods of time in the humid environmentsof moundsshould be an effective wav to causedisease and to target specificS. invicta colonies. MATERIALS AND METHODS Beatnteria bassiana (ARSEF#2484) was grown in shaking (100 rpm), sterile Sabouraud'sdextrose broth with lo/o yeast extract (SDBY) for five to six days at 24"C (Bextine and Thorvilson20l2b). After incubatiorl the contentsofthe flasks were strained through white muslin cloth so that mycelia were separatedfrom this broth. The separatedmycelia were mixed with a lo/o sodium alginate solution (2.5g of sodium alginate [Bioserv, Frenchtowr! NJ] mixed with 10.0m1of 95%oethanol and then addedtosterilewater).Myceliawereaddedataruteof3T.}gofwetmyceliaperl00mlof sodium alginatesolution The suspensionwas then mixed in a blenderwith 2.0g of wheat bran (Hodgson Mill, Inc. Teutopolis, IL). An uhraviolet-reflective, orange dye was incorporatedinto the fungal matrix so removal of the dried, alginate,B. bassianamatrix by ants oouldbe more easilyobserved. Woodencraft sticks(11.5cm x l.0cm x 0.2cm)and woodeq suryeyor's marking stakes (45cm x 2.5cm) were coated with the B. bassiana alginate suspensiorgdipped in 0.25M aqueous calcium gluconate sohrtion to gel tlre suspension,and allowed to dry (38"C). Alginated mycelia adh€redtightly to the sticls and stakes. For all batchesof B. bassiana-coatedsticks or stakes,a fungal samplefrom a stick or stakewas placed in a petri dish with sterilized filter paper (No. I Whatman),moistened with sterilize4 RO water, and incubatedat25"C to test whetherthe fungus would activate and produce conidiophoresand conidia. This precaution also ensuredthat the fungal ooatingwas not contaminated.Once B. bassianahydrated and producedconidi4 the batch was usedin experimentaltrials. 1997 Fungal Stick-TreatedMounds. Thirty S. invicta moundswith colony ratings of 25 (with brood and>50,000 workers) (Harlan et al. 1981,Lofgren and Williams 1982) were locatedon a plot of pasturelandin CassCounty, TX. Fifteen moundswere randomly selectedfor treatmentwith wooden craft sticks coatedwith B. bassiana. Three craft sticls coatedwith B. bassianawere insertedinto eachnpund. Craft sticks were placeddircctly into moundsleaving only 2-3cm aboveground level. No sticks were insertedinto fifteen, randomly selectedmounds of the control treatment. All nnunds used in this experiment were flagged, numbered, and tracked for six weeks. Changes in mounds, such as movemcnt, vacancy, and colony rating, were recorded. The appearanceof a similarly sized mound within 1.0m was consideredas movementof the samecolony. At the end of the experiment,the sticks were recoveredand observedfor mycelial growth. 1998 Tenth-HectareFungal Stick Treatnents. Ten 0.1-ha circles were randomly assignedtreatments. In five circles, all moundswere treated with two craft sticks coated with B. bassianaon 20 March. Each craft stick was placednear the centerof a mound and pushedin coqletely. All colonieswithin the other five circles were treatedwith two craft sticks tbat were not coatedwith B. bassiata. 48 Colony ratings were recorded ev€ry two weeks for an 8-week, post-treatn€nt period at every mound located within each 0.1-ha circle. Mouds with no activity were given a colony rating of 0. Bait cups were placed next to S. invicta moundsto collect individuals for evaluation of B. bassiana infection PercentsB. bassiatw infection that accumulatedin anls after 10 days in the labor*ory were wed to calculate means for statisticalcomparisons of treatments. 1998Individual Fungal StickTreatments. On I May 1998, 40 S. invicta mounds with colony ratings of 25 were randomly treated with either craft sticks coated with B. bassianaor craft sticks with rc B. bassiana. fuch mound was treatd with three craft sticks placed near tle center of the mound and pushedcompletely into tlre mound. A numberedsurveyor's.stake was placednext to eachmound for identification At 14 and 28 days, each colony was rated and S. irwicta sampleswere collected to observefor fungal growth in the laboratory. Mean percentsof accumulatedinfection of ants were calculated for statisticalcomparisons. Individual Fmgal Stake Treatments. With th€ successof the fungus-ooatedcraft sticks, an evaluation of larger stakes coated with B. bassiana was designed. The hypothesis was that with more fungus being introduced into mounds, more colony mortality would be observed"Numbered, sunreyor's wooden stakes for identification were placednext to ten rnoundswith colony ratings of 25. Each mound was randomly assigned one of two treatments. Five moundswere eachtreated with one, 45-cnr,wooden stake that had beencoated with B. bassiana. The remaining moundswere treatedwith 45-cm stakes that had not beencoated. At two and four weeksafter treatrnent,each mound was colony rate4 and S. irwicta sanry|aswere collectedto observefungal growth in the laboratory. In order to determineif the introduction of 8. bassianawas a mortality factor in all trials, S. invicta mdividuals were brought back to tle laboratory for observationof firngal infection For surface sterilizatioq ants were placed in l0olo Clorox@(5.25% sodium hypochlorite; Clorox Co., Oakland,CA) for l0 secondsand then flushed with water. Ants were tlrcn placed in petri disheson sterile fiher paper and observed. When fungal Srov{th was found, fungi were mountedon a microscopeslide and identified. B. bassianawas ro- isolated,grown on Sabouraud'sdextrose agar, and identified again. Within each treatment in all experiments,nrcan colony ratings were calculatecl. Analysis ofvariance (ANOVA) was usedto d€terminedifferences (critical P-value = 0.05) in mean colony ratings or percent infection between treatnrents(SAS 1996). Least Significant Differences(LSD) were usedto sepamtemean differences. RESI.]LTS 1997 Fungal Stick-TreatedMounds. After eight days, ,8. bassianaapplied using -- the craft stick methodcaused significant reductionin colony rating (F : 19.I ; df =1,28;P 0.0002; Table 1). At 32 and 5l days post-treatment,mean colony ratings were also significantlydifferent (F = 9.6;df : 1,28;P:0.0045, andF = 20.9;df :1,28; P:0.0001, r€spectively). On day 51, 93o/o(l4ll5) of S. inviaa colonies treated with B. bassiana- coatedcraft sticks were inactive (rating :0) comparedto 27o/o(4/15) ofcontrol mounds. 1998Tenth-Hectue Frorgal StickTreatments. The meanratings of coloniestreated with fungal sticks were lower than the control (sticks with rc B. bassiana)after 14 days(F :27.5,df=1,457;P<0.001;Table2). Thetrendofsignificantlydecliningcolonyratings in the S. invicta ooloniestreated with 8. bassiana-coaledcraft sticks continued;whereas, ratings of control colonies remainedfairly constant(Table 2). At 56 days, differencesin colony ratingswere highly significant(t :223.0; df : 1,388;P < 0.0001). Surprisingly, no ants collested from S. irn'icta firrtllrrdsin any of the treatmentsdeveloped B. bassiana 49 infections in the laboratory after being swface sterilized and placed in petri disheswith moistend sterile filter paper. TABLE 1. Mean S. invicta C.alonyRatings after Application of B. bassianaAlginate Craft Sticks. 9 July- 19 September1997. (BextineFarms, Cass Co., Texas). Mean colony rafing" Davs post-tredrnent Treatmentb 5l Fungal sticks 25.0a 6.0a 7.6a l.3a Control 25.0a l9 3b l9 3b l7 0b " Means followed by the sameletter within a column are not significantly different (ANOVA$LSD, P>0.05). o Three^B. bassiana-coverd craft sticks or no sticks applied. Fifteen moundsper treatment. TABLE 2. Mean S. irwicta C-obnyRatings after 0.l-ha Individual Mound Application of B. bassianaAlginate Craft Sticks. 20 March - 15 Irday1998. (Ber.tineFrms, Morris Co., Texas). Mean colony rating" Davs oost-treatnrent Treatmentb 0 14 28 42 56 Fungal sticks 19.9a 16.6a 12.0a 8.6a 7.5a Control l9 3a 2O2h 20.0h 19.5b 20,5b " Meansfollowed by the sameletter within a column are not significantly different (ANOVA" LSD, P >0.05). b Two 8. bassiana-coverdcraft sticks ortwo craft sticks with no 8. bassiana. Five 0.1-ha circles for eachtreatment. 1998Individual Fmgal Stick Treatnents. Becauseall S. irwicta colonies chosen for this trial had initial ratings of 25, no differencesat tbe time oftreatment were detected (Table 3). After 14 days, ratings of S. ilwiaa colonies treatd with B. bassiana+natd stickshad declined significantly (F :21.1; df = 1,38;P < 0.001). At day28,65% (13120) of S. invicta moundstreated with B. bassiana-coatedcraft sticks were renderedinactive (rating : 0) comparedto 5% (l/20) ofthe control mounds. TABLE 3. invicta Colony Ratings after Individual Mound ApplicationOf .8. bassiana Aleinate icks. Mav 1998 Monis Co.,Texas ratin$ Davs nost-treatrnent Treatmerrfb 0 14 28 Fungalsticks 25.0a 9.7a 3.2a Cnntrol 25.0s. 22.1b 18.2b " Meansfollowed by the sameletter within a column are not significantly different (ANOVALSD,P>0.05). b ThreeB. bassiana-oovercdcraftsticks or thee craft sticks without a B. bassianacnaffury. Trventy nrcundsreceived each treatment. After 14 days,99 of 190 individual ants collected from B. bassiana-tratedmounds developedB. bassianainfections after l0 days in the laboratory (Table 4), and infued ants were collected from 16 of 20 sampledcolonies. No ants (01219)from control mounds 50 developedfungal infeclion After 28 days, 461167errrts were infecte4 and infued ants *o" *ll""t"d-from 12 of the 20 sampledmounds. No infected ants were collected fiom control mounds(0 | 206). TABLE 4. Mean PercentAccumulated Infection caused by B. bassianaafter l0 Daysin the Laboratoryfrom Field-CollectedS. invicta. May/June1998. (BextineFarms, Morris Co..Texas). Mean percentaccumulafed infection' Davs Dost-treatment Treatmentb 14 28 f'nngal sticks 52.1a 27.5a Cleansticks 0.0b 0.0b " Means followed by the sameletter within a column are not significantly different (ANOVA' LSD, P > 0.05). Ants were held in petri disheswith moistened,sterile filter Paper. b ThreeB. bassiana-caverdcraft sticks ortlnee craft sticks without tB. bassianacoatmg per mound. Indivi&nl Fungal Stake Treatments. The treatmentof S. invicta colonieswith 45- cm stakescoated with B. bassianaproved to be effective (Table 5). After only 14 days, 80o/oof rnoundstreated with B. bassianawere renderedinactive as comparedta 0%oof control mounds. Mean colony ratings were significantly different betweentreatmentb at bothpost-treatment times (F :98.8; df : 1,8;P < 0.001,afrF :42.1, df : 1,8;P < 0.001, respectively). One fungus-treatedmound survived to 28 days and had a reducedcolony rating of 5 (<100 workersbut with brood). After 14 days, 83.6% of ants collected from fungal-treatedmounds developedB. bassianainfections when held for l0 days in the laboratory$able 6). Infected ants w€re collected from five of five sampledcolonies. After 28 days, 19 of 53 ants were infected from tbree of fwe sampledcolonies. No B. bassiana-infectedants w€re collected from control mounds TABLE 5. Mean .S. invicta Mound Ratings a1ftff Application of ,8. bassianaAlginate Stakes.May 1998(Bextine Farms, Morris Co.,Texas). f)avs rnst-treetrneni TreatmenP l4 28 Fungal Stake 25.0a l.3a l.3a Clean Stake 25.0a 22.5b 20.0b 'Means followed by the sameletter within a column are not signifioantly different (ANOVA LSD,P>0.05). b One woodenmarking stake(45cm x 2.5on) coatedwith B. bassianaor one stakenot coatedwith B. bassiara. Five moundsreceived each treatment. 5l TABLE 6. Mean PercentAccumulated Infection Causedby B. bassianaafter l0 Days in the Laboratoryfrom Field-CollectedS. invicta. June 1998. (BextineFarms, Morris Co., Davs post-treatrnpnt Treatmentb Fungal stake 83.6a 43.la Clean stake 0.0b 00h " Means followed by the sameletter within a column are not significantly different (ANOVA, LSD, D0.05). b Woodenstakes (45cm x 2.5cm) coatedwith B. bassianaor stakesnot ooatedwith B. bassiana DISCUSSION The placementof immovable objeAs coated with B. bassiota alginate slurry was an effective method of pathogen delivery to red imported fire ant colonies in mounds. Unlike alginate pellets that must be retrieved by foragersand delivered to a colony, ants were not able to remove the inoculum sourc,efrom the mound; therefore,the fungus was able to re-hydrateand produceinfective conidia (Bextine and Thorvilson 2002b). We were initially concernedthrt S. itwicta colony movementin responseto fungal stick or $taketreatment would negatethe application of the pathogen However, mound movement was not observedin this experiment. Also, large amounts of mycelia were deliveredmaking ant escapewithout infection diffrcuft. Becausethe stick or stakewas placedbelow the soil surface,little chancefor above ground insegt contact with the inoculum existed. Also, undergroundapplication may protect the pathogen from harmfirl uhraviolet radiation. B. bassiana is a poor soil competitor (White 1995); therefore, we would not expectthe fungus to persist in soil for long periods. For these reasons,non-target arthropods would not likely be adversely affected by B. bassiana,and S. irwicta would be speoifically targetedin areaswhere they dominateground fauna The delivery method for this biological control agent was effective and targeted specific S. invicta colonies. Mortality of colonies, rather than just reduction of colony rating, indicatedthat thesetreatments may be useful in eradicationof tlre pest from furite areas. Furthermore,application oftle sticks and stakeswas easy,and the nontoxic nature to humans of the entomopathogenicfimgus makes it especially safe for use by homeowners. In this study, we have developeda B. bassianadelivery systemthat was easyto apply, targeted specific S. invicta colony mounds, and allowed extended S. invicta exposurebecause ants could not remove the inoculum source. The method developedin this work may be applicableto homeowneruse of this safe and effective biological control agent. ACKNOWLEDGMENT This research was funded by the Texas Imponed Fire Ant Research and ManagementProject. This manuscript representsa portion of the senior author's MS. Thesis, Department of Plant and Soil Science,and is contribution T-4-536, College of Agricultural Sciencesand Natural Resources,Texas Tech University, Lubbock. 52 LITERATTIRE CITED Be*ine, B. R 1998. Application of Bearmeriabassiana alginate pellets as a biological conhol strategy against Solenopsisinvicta. MS. Thesis. Texas Tech Univenity, LubbochTexas.ll3p. Bextine, B. R., and H. G.Thorvilson2Dl2a. Monitoring Solmopsis iwicta (IIymetnoprai Formicidae) foraging with peanut oil-baited, [fV-reflective Beaweria bassiana alginatepellets. Southwest.Entonrol. 27: 3l-36. Bextine, B.R. and H.G. Thonrilson. 2002b.Field application of bait-formulatedBeaweria bassiona alginate pellas for biological control of the red imported fire ant Qlymenoptera:Formicidae). Erwiron Entonol 3l: 746'752. Broome, J.R 1974. Microbial conhol of the imported fire ant, Soletwpsisrichteri (Forel) (Hymenoptera: Formicidae). PtlD. dissertation, Mississippi State University, MississippiState, MS. 661 p. Harla& D.P.,W.A. Banls, H.L. Collins,and C.E. Stringer.1981. Large areatests of AC 217300 bait for control of imported fire ants in Alabama, Louisiana and Toras. Southwest.Entomol 6: 150-157. Lofgren, C.S. and D.F. Williams. 1982. Avermectin Bla highly pot€ntial inhibitor of reproductionby queensof the red importd fire ant (Hymenoptera:Formicidae). J. Econ Entornol 75: 798-803. Oi, D.H., RM. Pereira,J.L, Stimac,and L.A. Wood. 1994.Field applicationsof Beaweria bassiana for control of tlre red imported fire ant Slymenopera: Formicidae). J. Econ EntomoL 87: 623-630. Pereir4 R.M., S.B. Alves, and J.L. Stimac. 1993a Growth of Beaweria bassianarn ftre ant nest soil with arnendments.J. Invert. Pad;llc.L62:9-14. Pereira, RM., J.L. Stimac, and S.B. Alves. 1993b. Soil antagonismaffecting the dos+ responseof workers of the red imported fire an, Solenopsisirwicta, ta Beanteria bassianaconidia J. Invert.Parhol.6l: 156-161. SAS. 1996.SAS Institute Inc., Cary, NC, Siebeneicher,S. R, S. B. Vinsoq and C. M. Kenerley. 1992. Infection of tlte red imported fire ant by Beatneria bassianathrough various routes of exposure. J. Invert. Pa,thol.59: 280-285. Stirrac, J.L., RM Pereirg S.B. Alves, and L.A. Wood. 1990. Field evaluation of a Brazilian strain of Beaweria bassiana for coffiol of red imported fire ant, Solenopsisiwicto, in Florida. 5th Int. Colloq. Invert. Pathol Stimac,J.L., RM. Perer4 S.B. Alves, and L.A. Wood 1993. Mortality in laborarory colonies of Solmopsis invicta Qlyneroptera: Formicidae) treated with Beaweria bassiana@euteromycetes). J. Econ.Entomol. 86: 1083-1087. White H. E. 1995. Alginate pellet formulatbn of Beauveria bassianapathogenic to the red importd fire ant. M.S. Thesis. Dept. of Plant and Soil Science. TexasTech University, Lubbock, Toras. 69 p. 53 vol,.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 MORTALITY OF RED IMPORTED FIRE ANTS, SOLENOP$SIWICTA, CAUSED BY CASTOR SEEDS, RINCINUSCOMMUNIS. Eli Boroda and Hadan Thorvilson Departmentof Plantand Soil Science TexasTech University Lubbock,Texas 79409 ABSTRACT Red imported fire ants,Solenopsis iwictaBuren(Hymenoptera: Formicidae),from different oolonieswere fed castorseeds as a sole food source. Soybeanseeds or honey and water were used as contol food featnents. Regardlessofthe form ofcastor seeds,red imported fire anb were killed. Furthermore,the po*sibility of genetic predispositionby individual colonieswas not evident as a factor ofdeath. INTRODUCTION The red imported fite ant, Solenopsisinvicta Bwen (Hymenoptera:Formicidae), was intoduced into the United Statesin the 1930's and has movod urestwardat ebout 198 krn per year. Activities of humans,including nu$€ry material movementand constnrction projects,aided the rapid spread(Vinson 1997). Red imported fire ans have caused many social, economical, medical, and environmentalproblems. S. irwicta stings to humanscause painful pusfirleson the skin, secondaryinfections, oosmetic damage and, occasionally, life-threatening anaphylactic shock, S. irwicta causeagrioultural problems, including lqiury to livestock, reduction of crop productiorl and damage to farm equiprnent. Beoauseof their mound4uilding behavior, S, invicta genemE costly damageto electioal and communicationsequiprnent and to traffic confol syst€ms. Wildlife problems cr€aied by S. irwicta include altering ecological balance, impacting endangeredspecies, md interfering with recreational hunting and fishing. Invasion and infest*ion ofreal estatehave reducedproperty values ftat has led to significant economicloss (Viruon 1997). Researchersat tle Instituto de Biociencias in San Paulo, Brazil, reported that mortalrty rates in a leaf-cutting ant.Attq serdensrubropilosa (Hymenoptera:Formicidae), increasedafter consumptionof castorleaves, Ricinuscotnmunis (Euphorbiaceae) (Hebling et al. 1996). In anotherstudy at the FederalUnivenity of Agriculture in Umuahla,Nigeri4 the oultivdion of castor planf in a mixed cropping syst€,mreduced soil nematode populationsby 90o/oand significantly increasedyields ofcassava and cocoyamby 290/oand 28%, respectively. Swerity of nematodeinfestations in tuben and corms were reducedas well (Ugbaja1997). 55 Researchen at Texas Tech University conducted confrollod, mixed crop experiments using castor and sunfloner as protective borders for cotton. Aerial photographsrevealed a more dense canopy in cotton, as well as in nearby sudangrass, when the crop was near castor comparedto a sunflower border. The hypothesiswas that ground-inhabiting organisms wer€ suppressed by castor materials (8.B., personal observations). The castor plant has sweral toxins that are deadly if ingestedby insects,humans, and other animals. Thesetoxins may be naturally releasedby the plant directly into soil, upon deathof the plant, or the dropping of its seeds. Ricin is a compoundconoentrabd in the endospermof castor s€eds,is absent from otlrer plant parts, and is one of the most poisonousproteinaceous substances known (Moshkin 1986). Ricin is composedof two highly toxic polypeptides,ricin A-chain and B-chain, that are held togetherby a disulfide bond. B-chains bind to complex galactosideson cell membranesof eukaryoticcells. After being engulfed, both chains are tansported through the oell and eventually to ribosomes where the A-chain depurinatesthe adenine found in 28S ribosomal RNA subunib in mammalian cells (Lord et al. 1994). A single ricin molecule can inactivate over 1,50O ribosomes(Olsnes and Saltvedt 1975). Ricining an alkaloid also found in oastorleaves, seeds, and seedhulls, is released into soil, is consideredan important maabolite that panicipatesin the synthesisof castor proteins,is a potential allergen,and is a preoursorof ricin @ukhatchenko 1986). Ricinine oausedthe death of tortricid caterpillrs (Lepidoptera: Tortricidae) but has a lessereffect on humans(Bukhatchenko 1986). Becauseof its molecularweight (16a.2),ricinine, alone, is not immunogenicto humans,but when attachedto a larger and more complex organic molecule such as a hapten, found in human and bovine sera, elicits an immunological response(Artyukhova et al. 1992). Castor is grown as a speoialty crop for its non-toxic oil that is used exte'nsivelyin the cosmetic industry, as a motor lubricant, and in Nylon-Il (a polyamide) production. Nylon-ll may be the most valuable use of the castor plant and its oil, especially in the manufactureof plastics (ICOA 1992). Western Texas may be an ideal environmentfor castor culture as an alternative crop for cottorl but toxicity of castor plant parts is a problem in cultivation and marketing. Also, castormay be valuable as a pest management tactic to control nematodeand insect pest populations. However, bio-indicator organisms with clOseproximity to soil and crops, such as ants, may be necessaryto monitor toxic materialsassociated with castor. The objective of this laboratorystudy was to determineS. invicta mortality causedby feeding and exposureto various castorplant parts. IvTATERIALSAND METHODS Colonies of S. iwicta were collected from separateTexas sites to reduce genetic similarity. Twenty S. invicta from separatecolonies were placedin individual plastic petri dishesand randomly assigneda castorfood or control treatnent Sevenseparate trials were conduotedusing castor seedsas a food source for S. irwicta (Table 1). The ricin-acptone treatmentconsisted of 0.5g of acetonepolvder (3-3.5mg ricin) per ml of de-ionizedwater' The food contol teatn€nt was one soybeanseed, except in the ricin-acetonepowder trial where honey in sterile, revene osmosis@O) water was the confiol food source. Water contol treatmentsconsisted of sterile, glass shell vials containing sterile RO waler and pluggedwith sterile cotton; no food sourcewas available to ants in this confol teatment. Each peti dish also had a vial containing0.5m1 of distille4 sterile water. Petri disheswere placed within resealableplastic bags into which cotton balls moistenedwith stenle water were placed to maintain humid conditions. Five replications (n:5 colonies) of eaoh treatncnt were accomplished. 56 TABLE 1. Castorseed Treatmentsoffered to so/enopsisinvicta in separateTrials. : I onewholesee4 crushed 2 orgwhole see4 germinated 3 ricin-acetonepowder dissolvedin water (3-3.5m9ricin Perml water) 4 onecdYledonfromgemrinatedseed 5 one rootfrom germinatedseed 6 one whole, water-soakedseed after 24tr' without seedcoaj 7 one water-soakedseed after 24h- without seedcoat. cnrshed Mortality counts of s. iwicta werc taken daily. Accryqv gf dead ant counts n'as = fi'on day 0-8; verified by also counting live ants (totat 20 ants). Time Period I y3s Time period B was frori day 18-19; Time Period C was from &y ll-22t post-treatment' Death was determinedby observationof the curling of the body and lack of responseto nigoio* tapping and gentle shaking of pet'i dishes. Counts wEre terminatedon day 22 errenif not all antswere dead. Proportions (p) of dead ants oo'rnparedto total ants wer€ calculate4 and mem perc€ntage'morAlitiii were calculated. Proportional data were arcsine'transformed (ANovA, critical P-value = 0'05), and i"..rnJpl before one-way analysis of variance (mean means *-" separated w Tukey-Kranrer HSD. Unfiansformed data p"*.ntug"*"ulnulated mortality) were reported in Table 2. Analyses were completed using JMP Start@Ststistics (SAS Institute2000)' RESI]LTS A}.ID DISCUSSION At the end of eachtime period and within eachcastor treafrn€nt mortality in castor treatrnentswas significantly gt"t t than those in control treatments(Table 2). The only exceptions*ere ir tials with whole, crushedcastor seedsand whole, germinatedcastor seedsat the end ofPeriod A in which mortalities in castortreafinentswere not significantly different from those in control treatnents. Some differencesamong S. invicta mortality in food control treah€Nils existedbut were not considered important No significant differences in mortality among water controls were detectedat the end of Time Period C (F = 1'1488; df = 6, Ai P = O3654). No differencesin mortality amongcolonies were detectedin any time period (F = 0.3764; il = 4,X)i P =O.8z3r5after Time PeriodC)r therefore,genetic predisposition of coloniesto castorwas not indicated. After eachtime perio4 mean accurnulatedmortality of S. irlicta amongthe seven oastor treahents were compared (Table 2), and significant differences among castor treatmentswere detected. Cter Time Period A, percelrtagemortality ranged between0 and 33.0%. Whole, crtrshedcastor seeddid not causedeath among S. iroicta; wlrcreas, : = = other castortreatments caused significantly greatermortality (F 3.0265; ff 6, 28; P 0.0208). Acoumulatedmortality causedby germinatedseed cotyledon was significartly = :6' greaterthanthat causedby ricin-acetonepowder after Time PeriodB (F 3.3591;df 1S: p = 0.0128). At the end of the experiment(Time Period C), accumulatedp€rcentage ant mortality in all castor feafinents was X0.0%. S. irwicta mortality in petri dishes containing castor cotyledons, soaked castor seeds without coats, and soake4 crushed : = castorseeds without goatswas grealerthan that ofricin-acatone powder (F 4.6OZg;at 6,27;P:0.0024). 57 TABLE 2. MeanPercentage Accumulated Mortality of solenopsisinvictaExposed to SevenCastor Treafinents and Controls. Mean PercentaeeAccumulaled Mortalitv * SD " o Time Period Tr€atnent Whole, crushedcastor seed 0.0+0.0aA 82.0+25.6aA8 82.0+25.6aA8 Soybeanseed 0.0+0.0a 0.0+0.0b 0.0r0.0b Watercontrol 0.0 +0.0a 6.0 *5.5b 6.0 r5.5b ti/ii;G;d;rn'ffi64ili;;'i;;A'."""".ii.ii'+Iti;it-'-""'6,i.b'*iii.a;Aii"" i(i.d;ri.4;a,E Soybeanseed 3.8L7.sab 3.8+7.5b 3.8+7.5b Honey in RO water t.0+2.2b 8.0+ 8.4b 9.0*8.9b -t'---_':__'_'-_:____water control 1.0+2.2b 6.0+6.5c 6.0 +6.5c Germinatedcastor root 23.0+11.5a8 77.0 LlS.2aAB 93.0+7.6aAB Soybeanseed 2.0+4.5b I1.7+5.8b 13.3+2.9b water control 1.0+2.2b 3.0+4.5b 6.0+6.5b Soakedcastor seed do seedcoat 32.0+20.8aB 81.0+9.6aAB 97.0+4.SaA Soybeanseed 3.0*6.7b 5.0*8.7b 6.0+10.8b Watercontrol 1.3+2.5b 1.3+2.5b Soybeanseed 0.0+0.0b 2.5+5.0b 3.8+7.5b Water control t.3*2.5b 1.3*2.5b 1.3r2.5b Means followed by the samelower easeletter within a castorreatnent in a cohmrnare not significantly different. Means followed by the same upper case letter within each column are not signifioantly different (ANOVA, Tukey-KramerHSD; P > 0.05). Arosine y'proportions-tansformeddata. Untransformeddata are presentedin Table 2. b A, initiation of experimentto day 8; B, day 18 or 19; C, day lg Ia day22 The results of all trials indicated significantly greater mortality of sequesteredS. invicta irlaastor heafinentscompaled to contols. In many cases,all anb from the separate colonies died. Ricin-rich tissues,such as the endospermand water-soakedcastor seeds, may cause gleater mortality of S. invicta. Admittedly, castor materials were the only treatment food souroesin this experiment and ants may not choose castor in a field sihration. Also, anb were in close contact widr castor in eachperi dish an4 undoubtedly, walked acrossthe materials,as they would if encounteredin soil. Future experimenbwill addressthe applicationof castormaterials to soil and exposureof ants. Nonetheless,clear indications exist that ant deathswere directly related to castor, regardlessofthe state in which it was presented,and ant mortality differed only in the ratesat which ants died. The slower deathrates in the whole, germinatedcastor seed and ricin-aceton+fed nials may be due to degmdation of food or ohemical changesdue to germination and to repulsion, respectively. As with the results from Brazil with leaf-cutting ants (Hebling 1995), data from this trial indicate that castoris also deadly ta S. invicta. Perhaps,castor may be used to repel and kill S. invicta as well as other ground-dwelling pests of agriculture. In addition, S. ituicta and other soil-inhabiting organismsmay be valuable bio-indicators of toxins applied to soil and crops,as in.bio-terrorism- 58 ACKNOWLEDGMENT TheauthorsthankJ.S.Armshongforhisreviewofanearlyversionofthis provided manuscript. The Texas Imported Fire Ani Researchand ManagernentProject and l'*A"g ein this study. This manuscriptis contribution T4-s23,Deparfinent of Plant Teoh Soil S-cience,College of Agriculturh Sciences and Natural Resources, Texas University, Lubbock, Texas. LITERATI.]RECITED G' Y' Stamm' 192' Production ertyufaova"' E. A., P. K. Yuldashev'T. M. Bagalii, and of Ifrs to ricinine fromthe casior-oil plant. chem. Natural comp. 28: 445-446- Bukhatcheiko, S. L. 1986, Ricinine: the atkaloid of castoroil. p. 8l'85. In Y' A. Moshkin,(ed). Castor. AmerindPubl. Co. NewDehli' 315p' Hebling M. J. 4., P. S. Maroti, O. C. Bueno,O. A. da Silva, andF' C' Pagrocca' 1996' loxic effeds of leavesof Ricinuscommunis (Euphorbiaceae) to laboratorynests of Atta sexdes rubropilosaftIymenoptera: Formicidae)' Bull' Entomot' Res' 86: 253- 256. InternationalCastor Oil Association. 1992. The chemistryof castoroil and it derivatives and their applications. Westfiel4 NJ. Lord, M. r., r,. rvr^Roberts,and J. D. Robertus. 1994. Ricin: strush[e, mode of action, andsome current applications. FASEB J. 8: 201-208' Moshkin"V. A. (ed.) 1986. Castor.Amerind Publ. Co., New Delhi' 315 p' Olsnes,d., and E. Saltvedt. 1975. Conformation-dependentantigenic determinants in the toxic lectin ricin. J. Immunol. ll4: 1743-1748' SAS Institute. 2000. JMP@Statistical Discovery Software,version 4. SAS Institute Inc., SAS CampusDrive, Cary, NC' intercrops on Ugbaja,- - R. A. E. 1997. Effecb ofcastor uil plant/cassava/cocoyam loil nematode populatiog crop infestation, and yields of component crops' Biol' Agricul. Hort. 14: 177'185. VinsoU SI S. |SSZ. Invasion of the red importedfire ant. ,Amer.Entomol. 4?:23-39. 59 vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 TIIE EFFECT OF INTERPLANTING OF NECTERIFEROUS PLANTS ON TI{E POPIILATION DENSITY AND PARASMSM OF CABBAGE PESTS Mohammed A. Al-Doghairi! and Whitney S' Cranshawz/ Departmentof Bioagricultural Sciencesand Pest ldanagement Colorado StateUniversity, Fort Collins' CO 80523 ABSTRACT Nectariferousplants were interplantedwith cabbageto examinetheir effects on the population density and parasitism of cabbagepests, including the imported cabbageworm GCD\4/),Pieris rapre (L.), cabbagelooper (CL), Triclaplusiani (Hubner), diamondback moth @BM), Plutella rylostell.a (L.), and cabbageaphid (CA), Brevicoryru brassicac (L.). Mean parasitism of ICW larvae by Cotesiaglomeraa (L.) and Lespesiasp. ranged from 6.84 te 28.59%in 1993and from 15.97ta 27.60Voin 1994. ICW larvaecollected from cabbageplants that were adjacentto Good Bug Blend plots in 1993 and buckwheat and vetch plots in 1994 had the highest parasitism. Mean parasitism of CL larvae by Parocloidzs montants (Cresson) and Voria galis (Fallq) ranged from 0 to 28.88% in 1993and from 0.01 ta l.99Vo in 1994. CL larvaecollected from plantsgrown adjacent to dill plots in 1993 and alyssum and control plots in 1994 had the highest parasitism. Mean percentageparasitism of DBM larvae by Diadegma insulare (Cresson)ranged from 6.18 to L9.l37oin 1993and from 9,43 to 14.94%in 1994. Highestparasitism of DBM was observedin larvae collected from plants adjacentto vetch, Good Bug Blend, and dill plots in 1993 and buckwheat, alyssum and control plots in 1994. Mean percentageof parasitized cabbage aplid, Diacretiella rapae (M'Intosh), ranged from 4.78 ta lI%. Cabbageplants that were adjacent to bucl$heat had the highest number of parasitized aphids. Our results add to those of previous research indicating that interplanting or intercropping may enhancea natural enemy's activity againstpest species. INTRODUCTION Although host insects provide the diet for developmental stages of insect parasitoids,supplemental foods providing amino acid and carbohydratesoften are needed, particularly for speciesthat are not entomophagousduring some stages. Adults of many hymenopterousparasitoids are known to feed on flowers, aphid honeydew, host body fluids and other food supplements(kius 1960). Also, many predatorsrequire alternative food sourcesto enhancetheir reproductionand increasetheir predation (Bugg et al. 1991). l/King SaudUniversity, Qassim Branch,P.O.Box237 Buraydah8199, Gassim, Buriedah, Saudi Arabia ? Department of Bioagricultural Sciences and Pest Management, Colorado State University, Ft. Collins, CO 80523 61 Such nutrients can be obtained from nectar and pollen provided from flowers of crop plants, or from flowers of weedsand/or wild flowers surroundingor within the crop field (Altieri and Whitcomb 1979). T\e availability of one or more of the above supplemental food sources to the adult of beneficial parasitoids and predators is essential for their survival and reproduction, and poor performance of such biological conhols can be attributed to the absenceor scarcity of suchfood sources(Foster and Ruesink 1984, kius 1960).. Interplanting of nectariferousplants to provide supplementaryresources in attempts to enhancenatural enemies of a particular pest or pest complex is useful in biological controls. Several flowering plants, especially those with shallow and exposednectar flowers (e.g., the Apiaceae family, such as wild carrot and dill), are usable by many beneficial insects (Altieri and Whitcomb 1979, Bugg et al. 1987). The Polygonaceae family contains several important honey plants (e.g., domestic and wild buckwheats lFagpynm esculennnnMoench and,Eriogonwn spp., respectivelyl common knotweed lPolygorurn aviculare L.l) that featureexposed easily accessiblefloral nectarpresented in small flowers. These features of exposedfloral nectar often prompt heavy visitation by bees,wasps, flies, andother 'saccharophilic'insects, including braconid and ichneumonid parasitoids(Bugg et d. 1987). The primary objectives of this study were to illustrate the potential of certain nectariferousplants to increasethe activity of beneficialpredators and parasitesof cabbage pest insectsby providing them with alternativefood supplementssuch as nectarand pollen upon which adult stages feed. In addition this study allowed determination of the parasitoid complexesassociated with lrpidoptera on cabbagegrown in Colorado. MATERIALS AND METHODS The study was conductedduring the summer of 1993 urd 1994 at the Colorado StateUniversity Horticulture Field ResearchCenter, Fort Collins, Colorado. The study plot had dimensionsof approximately133 m in the east-westaxis and 125m in the north- south axis. The land was preparedand the plots was laid out and planted in May. Plots conained the following interplanting Eeatments: buckwheat, Fagopynnn esculentum 'Good Moench;common vetch , ViciasathtaL.; Bug Blend'; alyssum,Lobularia maitinw L.; dill, Anethwngraveolens L.; and a fallow plot as a control. 'Good Bug Blend' is a mix ofannual andperennial plants including: crurot, chervil, coriander,clovers (crimson, white, rose),nasturtium, parsley, alyssum and yarrow (PeacefulValley FarmSupply, P.O. Brx22@, GrassValley, CA 95945). The above flowering plants were chosenfor this study becauseof their ability to attract beneficial natural enemiesand some other insects (e.g., bees) and their ability to provide easily available nectar and pollen to their visitors. Treatmentswere separatedby 1.2-m plots planted with sweet corn as buffers. Rows were oriented north to south. Individual blocls were dispersedthroughout the field and separatedby different crops. Plots were regularly cleanedofweeds !o prevent confoundingeffects on natural enemies. All maintenanceoperations were made at least a day prior to sampling so that disturbed pests would have time to return to the plots. On the adjacent cabbage plants, populations were monitored for pests and percentageparasitism of key pests. Samplesfor all plots were taken on the sameday, weather permitting. For each sample, a number of cabbageplants were inspectedand records kept of the numbersof eggsof imported cabbageworm(ICW), Pieris rapae (L.), and cabbagelooper (CL), Trtchoplusia ni (Hubner), the number of larvae of ICW, CL, and diamondbackmoth (DBM) , Plutelln rylostella (L.), and the numberof parasitizedand 62 unparasitizedcabbageaphids(CA),Brevicorynebrassicae(L.).Late-instarlarvaeoflCW, Ci, and DBM were collected and transferredto the laboratory for rearing and determina- tion of percentageparasitism and the natureof the associatedparasitoid complex. I'arrre ofeach pest were reared in the laboratory in pefi dishesand regularly fed fresh cabbage leaves to prevent starvation that may result in eady pupation and cocoon construction. Ianvae were fed and maintaineduntil they died or pupatedor until Earasitoidsemerged. In the 1993 study, individual blocks consisted of two rows; cabbage (cv. 'CopenhagenMarket'), transplantedon 8 June in 40-cm in-row spacing, in one and the flowering plant treatments,seeded on 19 May and alyssumtransplanted on the sameday, in the other (12-m long plots). The statisticaldesign employed was randomizedcomplete block design, with five blocls each containing five treatmentplots and a fallow control plot. Daa were analyzedusing 2-way Analysis of Variance (PROC GLM) (SAS Institule 1985) to compare mean counts of eggs, lawae per plant and mean parasitism between treatmentsin different blocks. A significance level of 0.05 was used for all statistical tests. In the 1994 study, some alterations were made in the experimental design. Individual blocks consistedof five rows; rows l, 2, 4, and 5 were plantedto cabbage, transplantedon 17 May in 40-cmin-row spacing,and the middlerow, i.e, the third row, was used for flowering plant treatments(12-m long), seededon 20 May. An exception was alyssum which was transplantedon 3l May. Sampling was done on three cabbage plants from rows 1, 2, 4, and 5 adjacent to the interplanting treatments. Rows immdiately adjacentto the interplantingswere sampledseparat€ly from the outsiderows. The primary aim of this study was to determine whether certain specific interplanting arrangementsmight affect activity of natural controls in relatively small plantings, typical of regional home/marketgardens. However, it must be recognizedthat, although efforts were made to minimize experimental artifacts by siting design, the relatively small plot size involved in this study doesraise issuesabout "edge effect" in the design. Somemigration betweenplots of highly mobile species(e.g., tachinids) would be expectedand small plot designsmay not be suitable for detectinginterplanting effects. Data were analyzd using split-plot analysis of variance (PROC GLM) (SAS institute 1985); treatment was the main plot factor, and row location (nearer to treatment or farther from treatment) was the subplot factor. A significancelevel of0.05 was used for all satistical tests. RESULTSAND DISCUSSION In the 1993 study, numbersof eggs and larvae of imported cabbageworm(ICW) werenot significantlydifferent between treatments (Iable 1) (F:2.06, P< 0.08; F=0.3'1, P>0.86,respectively).However,treatmentshadasignificanteffectonparasitismoflCW larvae by all parasitoidspecies combined (Iable 1) (F=3.97, P<0.003). ICW larvae collected from cabbageplants that were adjacentto Good Bug Blend plots had significantly higher parasitism than larvae collected from plants adjacentto alyssumand control plots. Numbers of eggs and larvae and parasitism of cabbagelooper (CL) by all parasitoids combinedwere not significantly differentbetweentreatments (fable 1) (F:1.40, P>0.23; F:L.44, P>O.22; F:1.85, P>0.11, respectively).Treatments had no significanteffect on the number and parasitism of diamondbackmoth (DBM) larvae (fable L) (F:1.74, P> 0.13; F:0.64, P > 0.66, reqpectively). In the 1994 study, treatmentshad no significant effect on the numbersofICW eggs and larvae (Iable 2) (F:0.74, P>0.59; F=1.24, P>0.29, respectively). However, treatments had a significant effect on parasitism of ICW larvae by the complex of 63 parasitoids(Table2)(F=2.97,P<0.01). Iarvaecollectedfromplantsthatwereadjacent to buckwheatand vetch plots had significantly higher parasitismthan larvae collected from plants adjacent to dill and control plots. Row location had no significant effect on the number of larvae and parasitismof ICW (F=1.98, P>0.16; F=0.43, P>0.51, respectively). However, row location had a significant effect on the numberof ICW eggs (F :7 .6L, P > 0 .006) . Treatmentand row location interaction had no signifrcanteffect on ICWeggsand larvae (F:0.23, P>0.94; F=0.16, P>0.97, respectively).Howwer, the interactionhad a significanteffect on parasitismof ICW larvae(F:3. 16, P< 0.008). TABLE 1. Mean numbers of eggs, lanae, and percentageparasitism of imported cabbageworm,cabbage looper, and diamondbackmoth, collected from cabbageplants in buckwheat,common vetch, Good Bug Blend, alyssum,dill, and control plots, during the 1993 study, Colorado StateUniversity Horticulture Field ResearchCenter in Fort Collins, Colorado." Plot Eggs Parasitism ImportedCabbagewonn Buclovheat 52.7 a 8.4a 19.7 ^b Vetch 57.7a 7.8a 20.8 ab Good Bug Blend 60.9 a 7.6a 28.6 a Alyssum 53.5a 7.7a 13.4 b Dill 5.7a 9.4a 14.6 ^b Control 72.5a 8.1a 6.8 b Cabbagelooper Buclovheat 0.9a 2.3a 23.8a Vetch 1.0a 2.8a 21.2 a Good Bug Blend 0.3a L.7a 15.9a Alyssum 0.5 a 1.3a 20.2 a Dill 0.3a 1.8a 28.9 a Control 1.2a 2.0a 0.0 a DianwndbockMoth Buclavheat 2.6a 11.0a Vetch 3.4a 17.7a Good Bug Blend 2.3a 18.3a Alyssum 5.4a 7.3a Dill 3.5a 19.1a ConFol 3.4a 6.2a " For eachspecies, means within a column followed by the sameletter are not significantly different (P=0.05) by SNK test. Meansbased on eight cabbageplants per plot for five replications and three sampling dates. Number of eggs and parasitism of CL by the complex of parasitoids were not significanfl y differentbetween treatments (Table 2) (F :0.7 3, P > 0.60; F =0.93, P > 0. 46, respectively). However, treatmentshad a significant effect on tlte number of CL larvae (table 2) (F=3.06, P<0.01). The number of CL larvae was significanfly lower in 64 cabbageplants that were adjacentto buckr*,heat,Good Bug Blend, and alyslum plots than to con-trolplots. Row location had no significant effect on the number of CL eggs and larvaeand on parasitismof CL larvae(F:0.56, P>0.45; F:3.01, P>0.08; F=2'04, p>0.15, respectively). Treatment and row location interaction had no significant effect on CL eggs and larvae and on parasitismof CL larvae (F=1.U2, P>0'40; F:l'77i P>0.11; F: 0.82, P>0.53, respectively). TABLE 2. Mean numbers of eggs, lanrae, and percentageparasitism of imported cabbageworm, cabbage looper, and diamondback moth, count€d and collected from cabbageplants in buckwheat,common vetch, Good Bug Blend, alyssum, dill, and control plots, for the 1994 study, at the Colorado State Univenity Horticulture Field Research Center in Fort Collins, Colorado." Plot Eggs Parasitism Imported Cabbaganorm Buclovheat 33.5 a 2O.3a 26.5 a Verch 40.9 a 22.4a 27.6a Good Bug Blend 38.6a 23.0a 23.4 ab Alyssum 32.8a 15.1a 17.6ab Dill 37.9a 2L.7a 16.0 b Control 34.8a 19.6a 16.8 b CabbageLaoper Buckwheat O.2a 0.7 a 0.0 a Vetch 0.5 a 1.0ab 0.0a Good Bug Blend 0.5 a 0.5a 0.0 a Alyssum 0.4 a 0.7 a I.6 a Dill 0.4 a 0.9 a 0.0 a ConEol 0.5 a 1.3 b 2.0a Dianondback Moth Buckwheat 0.5 a 14.0a Vetch 0.5 a 9.9a Good Bug Blend 0.5 a 9.5a Alyssum 1.0 b 14.9a Dill 0.3 a 9.4a Control 0.5 a 14.0a . For eachspecies, means within a column followed by the sameletter are not significantly different (P=0.05) by SNK test. Means basedon twelve cabbageplants per plot (three plants per row) for five replications and six samplingdates. Treatments had a significant effect on the number of DBM larvae (Iable 2) (F=2.87, P<0.01). Number of DBM larvae were significantlyhigher in the cabbage plants that were adjacentto alyssumcompared to other treatments. Ilowever, parasitism of DBM lawae was not significantly different among treatments (Table 2) (F:0.50' P>0.77). Row location had no significant effect on the number of larvae and parasitism of DBM (f:1.86, P>0.17; F=3.17, P>o.W, respectively).Treatment and row location interaction had no significant effect on the number of larvae and parasitism of DBM 65 (F=0. 70, P > 0.62; F =0.44, P > 0.8 l, respectively). Treatmentshad no significant effect on the numberofparasitized and unparasitized cabbageaphids(CA) (Table3) (F:l.2l,P>0.30; F:L82,P>0.10, respectively).Row location also had no significant effect on number of parasitized and unparasitizd CA (F=1.04, P>0.30; F=0.35, P>0.55, respectively).Treatment and row interactionhad no significant effect on the number of parasitized and unparasitized CA (F=0.89, P> 0,48; F :0.43, P > 0.82, respectively). TABLE 3. Mean numbers of parasitized and unparasitizedaphids counted on cabbage plantsin buclrvheat,common vetch, GoodBug Blend, alyssum,dill, and control plots in the 1994 study at the Colorado StateUniversity Horticulture Field ResearchCenter in Fort Collins, Colorado." Plot Parasitizedaphids Unparasitizedaphids Buckwheat 11.0a 13.2 a Commonvetch 6.5a 6.7a Good Bug Blend 4.8a 4.3a Alyssum 6.0 a 3.4a Dill 5.2a 3.5a Control 5.7a 3.7a " All meansare not significant(P:0.05) by SNK test. Meansbased on twelve cabbage plants per plot (three plants per row) for five replications for six sampling dates. Two larval parasitoids, Cotesia (:Apanteles) glomeraa (L.) (Hymenoptera: Braconidae) and l*spesia sp. (Diptera: Tachinidae)emerged from ICW larvae collected in the field. C. glomerata femalesinsert eggsinto ICW first-instar larvae, and 16-52wasp larvae develop inside eachcaterpillar. They then emergefrom fifth-instar ICW larvae to spin yellow to orangecocoons in a group looselyattached to the host (Mahr et.al. 1993). Lespesiasp. femaleslay an egg on the body of ICW larva, the egg hatchesand the larva then bores into the caterpillar. Developmentoccurs within the laryal host and emerges from the pupa (Mahr et.al. 193). Overall parasitism of ICW larvae by the above parasitoidsranged from 6.8 to 28.6voin 1993and from 16.0 to 27.6Voin 1994(Iables L and 2 respectively). ICW larvae collected from cabbageplants that were adjacentto GoodBug Blendplots in this 1993study and vetch andbuckwheat in this 1994study had the greatestparasitism. Two larval parasitoids, Patocloides montanus (Cresson) (Hymenoptera: Ichneumonidae)and Voria ruralis (Fallen) (Diptera: Tachinidae),emerged from CL larvae collected in the freld. One or more larvae of V. ruralis develop within the host and emergefrom fully grown CL lawa (Mahr et.al. 1993). Overall parasitismof CL larvae by the previously mentionedlarval parasitoidsranged from 0 to 28.9% n 1993 and from 0.01 to 2Vo in 1994 (Tables I and 2 respectively). CL larvae collected form cabbage plants that were adjacent to dill plots in 1993 and alyssum in L994 had the highest parasitism. Diadegma insulare (Cresson)(Hymenoptera: Ichneumonidae) was the only parasitoid recoveredfrom DBM larvae collected in the field. D. insulare parasitizesthe later instar DBM larvae, but it emergesfrom the host pupa (Mahr et.al. 1993). Overall meanparasitism of DBM larvaeby D. insulare rangedfrom 6. 18 !o 19.13%in 1993and from 9.43 to 14.94%n 1994(Tables I and2, respectively).DBM larvaecollected from cabbageplants that were adjacentto vetch, Good Bug Blend, and dill in 1993 and 66 buclopheatand alyssum in 1994 had the highest percentparasitism among treatments. Diaeretiella rapae (M'lntosh) (Hymenoptera:Braconidae) was the only parasitoid recovered from parasitized aphids collected in the interplanting treatmentsadjacent to cabbageplants. FemaleD. rapae depositan egg in the aphid nymph and after egg hatch "mummy" the wasp larva consumesall body contentsof the aphid resulting in a (Mahr et.al. 1993). Results from this study showedno apparenteffects of treatmentsstudied on the densitiesof eggs,larvae, and on the parasitismof cabbagepest insects. This is consistent with results of other studies comparing various pest insects in monocultures and polycultures(Sheehan 1986, Laub and Luna 1992,Pavuk and Stinner 1992, Pavuk and Barrett 1993). Although there were no consistentdifferences in parasitism of cabbage pests betweentreatments, a trend for greater parasitismin plots interplanted with flowers than the controf plots was observed(Tables 1, 2). The higher parasitismin more diverse plots agree with results from other studies comparing parasitism in monoculturesand polycultures(Sheehan 1986), but they contrastwith the results of Horn (1987), who reported higher parasitismof DBM in tilled collard plots than in collard plots with weeds. Talekar et al. (1986) reported a similar trend, with no apparent significant differencesofnumbers ofDBM larvae wheri cabbagewas intercroppedwith severalother plant speciescompared with cabbagemonocultures. However, Grossman(1993) observed thatplots with vetch and bare ground plots had higher numberof CL larvae than plots with rye. Grc,ssmanalso reported no significant differencesin numberof CL eggsamong rye, vetch, and bare ground plots. Irius (1967) reported that the incidence of parasitism by hymenopteranparasitoids in orchards, against tent caterpillar and codling moth, was increaseddue to the rich undergrowth of wild flowers comparedto poor floral under- growth. Moreover, Tonhasca(1993) reported that natural enemiesof soybeanherbivores were more abundantin polyculture than in monocultureplots. In the studiesreported here, there generally were no overall apparentstatistieally significant differencesdue to interplanting treatments. Thus, there was no clear evidence that control ofphytophagous insectswas enhancedby the interplanting regime used. Also, natural enemy densitiesand responseto interplanting treatmentswere generally variable from year to year. ACKNOWLEDGMENT The authors wish to express their gratitude and appreciation to Dr. Boris C. Kondratieff for his patience and understandingand continual willingness to provide assistanceand suggestion. Further appreciationis due Dr. Phillip L. Chapmanfor his statisticalconsulting, and to Dr. E.E. Grissell and Dr. P.M. Marsh of the USDA-ARS SystematicEntomology Labotatory for identifications of parasitic Hymenoptera. Thanks also goes to Casey Sclar, Dayna Cooper, Thomas Eckberg, Simone Milsapp, Abra (Holtzer) Houchin, and Nina Pokriots for assistanceduring the freld work and collection of data. LITERATURE CITED Altieri, M.A., andW.H. Whitcomb.1979. The potentialuse of weedsin the manipulation of beneficialinsects. Hort. Sci. 8: 12-18. Altieri, M.A., andW.H. Whitcomb.1980. Weed manipulation for insectpest management in corn. Environ. Manasement4:483489. 67 Bugg, R.L., L.E. Ehler, andL.T. Wilson. 1987.Effect of commonknotweed (po$gonun aviculare) on abundanceand efficiency ofinsect predatorsofcrop pests.Hilgardia 55: 1-52. Bugg, R.L., J.D. Dutcher, and S.C. Phatak.1988. Cool- and warm-seasoncover crops in the pecan groves of southem Georgia: Management for soil fertility and biologicalcontrol. Northern Nut GrowersAssoc. 79: 53-58. Bugg,R.L., F.L. Wackers,K.E. Brunson,J.D. Dutcher,and S.C. Phatak.1991. Cool- seasoncover crops relay intercroppedwith cantaloupe:influence on a generalist predator, Geocorispuncrtpes (Hemiptera: Lygaeidae). J. Econ. Entomol. 84:408- 416. Foster, M.A., and W.G. Ruesink. 1984. Influenceof flowering weedsassociated with reduced tillage in corn on a black cutworm (*pidoptera: Noctuidae) parasitoid, Meteorus raDenr (Neesvon Esenbeck).Environ. Entomol. 13: 664-668. Grossman,J. 1993.Fighting insects with living mulches.The IPM Practitioner.XV: 1-8. Ilorn, D.J. 1987. Vegeational backgroundand parasitism of larval diamond-backmoth on collards.Entomol. Exp. Appl. 43:3N-303. Laub, C.A., and J.M. Luna. 1992.Winter cover crop suppressionpractices and natural enemiesof armyworm (Lepidoptera:Noctuidae) in no-till corn. Environ. Entomol. 2l:41-49. Irius, K. 1960. Attractiveness of different foods and flowers to the adults of some Hymenopterousparasites. Can. Entomol. 92: 369-376. Irius, K. 1967. Influence of wild flowers on parasitism of tent caterpillar and codling moth. Can. Entomol.99:444-446. Mahr, S.E.R., D.L. Mahr, and J.A. Wyman. 1993.Biological control of insectpests of cabbageand other crucifers. North Central Regional Publication. 47L. 55 W. Pavuk, D.M., and B.R. Stinner. 1992.Influence of weedcommunities in corn plantings on parasitism of Ostrinia nubilalis (Lepidoptera:Pyralidae) by Eriborus terebrans (Hymenoptera;Ichneumonidae). Biol. Control. 2:312-316. Pavuk, D.M., and G.W. Barrett. 1993.Influence of successionaland grassycorridor on parasitism of Plathypenascabra (F.) (Irpidoptera: Noctuidae) larvae in soybean agroecosystems.Environ. Entomol. 22: 541-546. Peaceful Valley Farm Supply. 1994. Tools and supplies for organic farming and gardening.P.O. Box 2209, GrassValley, CA, 95945, l2l pp. SAS Institute. 1985. SAS Users Guide: Statistics,Version 5th edition. SAS Institute, Cary, N.C. Sheehan,W. 1986. Responseby specialistand generalistnatural enemiesto agroecosystem diversification: A selectivereview. Environ. Entomol. 15: 456461. Talekar, N.S., S.T. Lee, and S.W. Huang. 1986.Intercropping and modificationof irri- gationmethod for the oontrolof diamondbackmoth, pp. 145-151./n N.S. Talekar andT.D. Griggs[eds.], Diamondbackmoth management: Proceedings of the First International Workshop. Asian Vegetable Research and Development Center, Shanhus,Taiwan. 471 pp. Tonhasca, A., jr. 1993. Effects of agroecosystemdiversification on natural enemiesof soybeanherbivores. Entomol. exp. Appl. 69:83-90. 68 vol.29 NO.l SOUTHWESTERNENTOMOLOGIST MAR.2004 EVALUATION OF CATOTIICCUSGRANDIS (HYMENOPTERA: PTEROMALIDAE) FOR BOLL WEEVIL' CONTROLIN NORTHEASTERNMEXICO E. Cortez-Mondac*,L.A. Rodriguez-del-Bosque,J. Vargas-Camplis, R, J. Coleman3, and J. L. Ley'va-Yflzqtrez" Instituto Nacional de InvestigacionesForestales, Agricolas y Pecuarias.Campo ExperimentalRio Bravo. ApartadoPostal 172,Rio Bravo, Tamaulipas,M6xico 88900 ABSTRACT Boll weevil cohortswere exposedin check and releasefields to assessthe mortality of boll weevil immaturesassignable to Catolaccusgrandis (Burks) and otJreragents tluough life table analyses.Field sites in Mexico were at Estaci6n Cuauhtemoc,Tamaulipas and Ebano, San Luis Potosi during late surnmer of 1999. Results indicated that Catolaccus grandis was the main boll weevil mortality faotor, primarily of third-instar larvae. Seasonal averagemortality of third instarswas higher at the Cuauhtemocrelease plot (74.1%) than at the Ebano releaseplot (64.30/o).However, overall mortality due to C. grandis parasitism was much lower at Cuauhtemocthan at Ebanoduring the crucial early period of boll weevil infestation and establishment,and the effect of the parasitoidswas not suffrcientto prevent severecrop damagein the Cuauhtemocrelease plot. In contast, higher mortality occuning at Ebano during the early infestation period (> 70y0 may have resulted in a slower population increase, lower overall weevil densities and percentagedamaged fruit. Al Ebano,satisfactory conhol of boll weevil was observedin the releasefield while the check required 1 I insecticide applications for boll weevil control. In the check plot at Cuauhtemoc,early seasoninsecticide applications for boll weevil control causedsufftcient natural enemymortality resulting in the needto contol SpodopteraexiguaHttbner INTRODUCTION The boll weettil,Anthonomus grandis Bohernan,is the most important insectpest of cotton in NortheasternMexico, not only becauseof yield lossesand insecticide confrol costs,but for its importanceand impact on the integratedpest management of the heliothins complex and other lepidopteransthat may be held in check by natural enemieswhen non- target insecticideinduced mortality is minimized. Researchon Catolaccusgrandis (Burks) has demonstated its potential as an augmentativebiological control agent for suppression of boll weevil in experimentaland commercialcotton fields (King et al. 1995,Coleman et al. 1996,Vargas-Canplis et al. 1998). The objectiveof this studywas to evaluatefield releasesof C. grandis in two NortheasternMexico locations. lColeoptera: Curculionidae. 'Current address:INIFAP, Campo Experimental Valle del Fuerte. Guasave,Sin., M6xico 81200. 'USDA/ARS/SARC,2413 E.Hwy. 83, Weslaco,TX 78596. aColegio de Postgraduados,IFIT. Montecillo, Edo. de M€xico, M6xico 56230. 69 MATERI,ALS AND METHODS Selectedfield sites were in nortleastern Mexico at two experimentstations of the Instituto Nacional de InvestigacionesForestales Agricolas y Pecuarias(INIFAP) locatedat Estaci6nCuauhtemoe; Tanraulipas; and Ebano;San Luis Pstosi. Plstswere plantedduring the filstwedcof August, 1999.Catolaccus grandis releaseplots were plantedto a Bt cotton (NuCOTON) and the commercial check was planted to Deltapine 50. The use of NuCOTON in the C. grandis releaseplots was to avoid insecticideapplication againstthe heliothinecomplex, which would affect parasitoidperformance. Plot size of check and releasefields were L2 and 4.8 hectaresat Cuauhtemocand Ebano,respectively. The parasitoids were produced at the Rio Bravo Experiment Station Laboratory usingboll weevil pupaefrom the APHIS BiologicalControl Center at Mission,Texas. Prior to release, female parasitoids were exposed to boll weevil larvae for two days in the laboratory.Catolaccus grandis were releasedtwice per week at the rate of 600 femalesper hectare.At the Cuauhtemocstation, 12 releaseswJre conductedbeginning September i7. At Ebano,14 releaseswere conducted beginning September 20. The releasefields were500 m away from check fields which were managed according to common commercial practicesfor the region (Table l). TABLE l. Insecticideapplications in Ebano,S.L.P., and Cuauhtemoc, Tam. 1999. Cuauhtemoc Date Insecticide" Date Insecticideo Checkfield 03-X parathionmet. A. grandis l5-Ix fipronil A. grandis 02-X parathionmet. A. grandis 2l-lx fipronil A. grandis 06-X parathionmet. A. grandis 27-V thiodicarb S. exigua 12-)( parathionmet. A. grandis 04-x fipronil A. grandis 2l-X parathionmet. A. grandis rz-x fipronil A. grandis 26-X parathionmet. A. grandis 18-X fipronil A. grandis 30-X parathionmet. A. grandis 22-X fipronil A. grandis M-XI parathionmet. A. gandk 27-X parathionmet. A. grandis lO-X parathionmet. A. grandis + l8-XI parathionmet. A. grandis metamidofos S. exigta 24-Xl parathionmet. A. grandis 01-XI parathionmet. A. grandis 23-)fJ fipronil A. grandis Catolaccusrelease field 16-X oxamyl Mirids No application I8.XI parathionmet. A. grandis 24-XI ion met. : Application rate (g A.L/ha) in all oaseswas as follows: parathionmet. = 750; oxarrryl 226; fipronil = 200; thiodicarb= 375; andmetamifofos = 600. After commencement of parasitoid releases, experimental plots were sampled twice weekly. Sample units were one square meter and sample size was eight and ten samples at Cuauhtemoc and Ebano, respectively. Information recorded included number of: a) undamaged squares and bolls on plants; b) botl weevil infested sqrvues and bolls on tho plant and ground; and c) squares and bolls damaged by other insect pests. All boll weevil infested forms were collected and retumed to the laboratory for inspection and 70 determinationof mortality factors. In addition, 100 squareswere randomly collected to estimatepercentage square damage by the boll weevil on each sampling date.Spodoptera exiguaHibner populationswere presentin Cuauhtemoc,and its damagewas also recorded. To estimate boll weevil generationalmortality, "open cohorts" were establishedto eonstruotlife tables (Morales-Ramoset al. 1995): Ten sqrmes irfested with'egg ur filst- inStf,f'boll'fii€bvil,as describedby Morales-Ramoset al. (1995),were attachedto a one- meter cord. Ten cords with infested squaf,eswere placed randomly on the ground beneath the cotton canopy twice per week during four weeks at Cuauhtemocand six weeks at Ebano.Each cohort was left in the field for two weeks.This allowed sufficient time for all mortahfy factors to occur or for adult weevils to emergefrom squares.The squareswere recoveredfrom the field and inspectedin the laboratory to determineif and at what life stage any mortality occurred, and the cause of the mortality according to the method describedby Sturm and Sterling (1986). At Cuauhtemoc,the first cohort was establishedon 6 October and the last 16 November (13 dates) while at Ebano the first cohort was established24 Septemberand the last 5 November (12 dates).Life table analysiswas used io measurestage specific mortality. The effect of parasitismby C. grandis and unexplained mortality were evaluated.by calculating stage and factor specific mortality rates (4") and indispensablemortality (1) according to Southwood (1978). Unexplainedmortality included the combination of desiccation,diseases, and mortality induced from feeding or venomization by adult C. grandis females. Thesetlree factors cannot be unambiguously identified from cohort samples (Morales-Ramoset al. 1995). The number of live "x" individuals initiating age age (lx) was calculated according to proceduresby Krebs (1e85). RESULTSAND DISCUSSION Cuauhternoc.Table 2 showsthe resultsof life table analysisfor all pooled boll weevil cohorts at Cuauhtemoc. Approximately 20o/omortality occurred in the egg stagein both check and releasefields, while survival of boll weevil from the egg to adult stagewas 78.4 and 13.9% in the checkand release fields, respectively. In the releasefield, 74 .lo/o mortality occurted in third-instar larvae, with 64.9% xtibutable to parasitismby C. grandis. For second-instar larvae and pupae, apparent mortality was approximately 12 and 28o/o, respecfively. Almost negligible mortality of larvaeand pupae was recordedin the check. Fig, l, shows the apparentmortality percentagesof boll weevil third-instar larvae due to parasitism by C. grandis recorded for each sample date during the evaluation at the Cuaulrtemocrelease plot. For the second cohort sample date, the apparent mortality dropped below 40%. It is very important that high percentagemortality be obtained by augmentativerelease of parasitoidsduring early seasoninfestation by boll weevil to limit pest population increase.After the last release(5 November), approximately 50%olarval mortality was rocorded until 12 November. Boll weevil damage in tle release plot exceededthe economic tbreshold beginning the fourth sample date until conclusion oI sampling (Fig. l, Table 3), while in the check plot, insecticide applications maintained damagebelow the economic threshold until the penultimatesampling date. In the check plot, damagingpopulations of S. exigua occrured (Table 3), possibly as a result of early insecticidesprays directed at boll weevil which eliminatednatural enemy regulation. Ebano. Resultsof life table analysisfor all pooled boll weevil cohortsat Ebano(Table 4) shows12-15% mortality occurred in the eggstage in botll checkand release fields, while survival of boll weevil from the egg to adult stagewas 83.4 and 27.5% in the check and releasefields, respectively. In the release field, 64.30/omortality occurred in third-instar larvae, with 62.2Yoattibfiable to parasitismby C. grandis. For secondinstar-larvae and pupae,the apparentmortality was approximatelyl0 and2.4%,respectively. In both release 7l and check plots, low levels of mortality caused by other parasitoids of third-instar boll weevil were recorded. In general, very low mortality of larvae and pupae was recorded in the check. TABLE 2. Life table analysis of boll weevil cohorts in Cuauhtemoc,Tam., from 6 October to 5 November 1999. Check field Cat o I ac cus release field Stage Lx" Nxb Ox' Ixd Lx" Nxb Qx" lxd Egg 887 1.000 19.4 18.9 899 1.000 l9.l 3.3 Instar1 U.M. 7rs 0.806 1.3 1.0 727 0.808 2.r 0.3 Instar2 U.M. 706 0.795 0.3 0.2 712 0.79r I1.8 1.9 Instar 3 704 0.793 1.0 0.8 628 0.698 74.r 36.6 U.M." 1.0 0.8 6.7 3.4 M.P.f 0.0 0.0 64.9 32.0 M.A.P.C 0.0 0.0 2.5 2.6 Pupae 697 0.785 0.1 0.1 t73 0.192 27.7 5.3 U,M.. 0.0 0.0 6.4 r.2 M.P.f 0.1 0.1 21.4 4.r Adult 696 0.784 125 ol-x is numberof live individualsstarting stage x. \x, is propotion of live individualsduring stagex. oQx is percentofindividuals dying during stagex. olx is tqe percent indispensablemortality occurring during stagex. tU.M. is unexplainedmonality (includesdesiccation, diseases, and host feeding). 'M.P. is C. grandis parasitismmortality. sM.a.p. is mortality by otherparasitoids. -+- Parasitism 100 + Damage/check +Damage/release 80 soo 40 20 0 o4.Xo6-X12.x15-x1g-x22-x26-X29.X02-X|05.x|09-X|12-X|16-X|19.X| Sample dates FIG. 1. Percentageboll weevil squaredamage in cotton and apparentparasitism ofthird- instarlarvae in cohorts.Cuauhtemoc, Tam. 1999' 72 TABLE 3. Percentage damaged squares in random and square meter samples at Cuauhtemoc,Tam. 1999. D/I00s" D/S m2n Adultsc D/100s" D/S m2t Adults" gv/b Date Bwb Awb B$/b AIMb gwb *wb Blvb Arilt *wb Bwt Arilb Check field Catol ac cus release field 04-x 000.00.000 t2 l 4.3 0.4 07-x 000.00,000 923.2 0.0 00 l1-x 000.00.000 l0 I 8.4 0.3 00 l4-x 500.40.000 t4 l t6.9 0.0 00 l8-x 500.00.000 33020.7 0.7 2l 2t-x 1034.33.102 21 0 19.3 0.0 00 25-)K 3 0 2.2 7.0 0 4 230 17.4 0.3 0l 28-X 000.92.500 r70 3.6 0.0 00 OI-XI 031.49.903 31 0 r5.6 0.0 30 04-XI 510.7r.702 25020.2 0.0 00 O8.XI 853.56.012 20031.8 0.0 10 II-XI 12 3 7.9 5.4 4 0 24030.0 0.9 00 l5-xI 18 3 10,6 8.2 6 4 40056.4 0.0 20 r8-)(I 47 7 12.6 5.9 6 6 53 l 58.8 0.5 4o "D/100 S= Damageper 100 squares;D/S m2= Damagedsquares per mt' oBW= BoU weevil; AW= Beet Armyworm. 'Adult - boll weevils in eight samplesites of I m'. TABLE 4. Life table analysis of boll weevil cohorts in Ebano, S'L.P. from 24 September to 5Noffi Check field Catol ac cus releasefi eld ffi Lx" Nxo Egg 1243 1.000 14.7 14.4 1196 1.000 r2.r 3.8 Instarl U.M. 1060 0.852 0.0 0.0 1051 0.878 0.3 0.1 Instar2U.M. 1060 0.852 |.2 0.6 1048 0.876 10.0 3.1 Instar 3 1047 0.942 0.8 0.6 943 0.788 64.3 49.5 U.M.. 0.2 0.2 1.9 1.5 M.P.f 0.4 0.3 62.2 47.8 M.A.P.g 0.2 0.2 0.3 1.0 Pupae 1039 0.835 0.2 0.2 337 0.281 2.4 0.7 U.M.. 0.2 0.2 2.t 0.6 M.P.f 0.0 0.0 0.3 0.1 Adult t037 0.834 "Lx is number of live individuals starting stagex' \$x is proportion of live individuals during stagex. "Qx is percentof individualsdying stagex. dI* ir th" percent indispensablemortality ocourring during stagex. tr.lr,t. is unexplainedmortality (includesdesiccation, diseases, and host feoding). tM.p. is C. grandis parasitismmortality. sM,e.p. is mortality by other parasitoids. 73 +Parasitism -+Damage/check. +Darmgey'release 2+rx 28-tx 01-x 26-X 29X o&.Xt05-Xt 09xt 12-xtlsxl Sampledates FIG. 2. Percentageboll weevil squaredamage in cotton and apparentparasitism ofthird. instarlarvae in cohorts.Ebano, S. L. P. 1999. The apparentmortality percentagesof boll weevil third-instar larvae attributable to parasitism by C. grandis for each sample date during the evaluation at the Ebano releass plot are shown in Fig. 2. More thanT}yo mortality of boll weevil third instarswas recorded in the first three sampledates. Then, mortality droppedto below 20%oon the fifth samplo before increasingin later sampledates again to more than 70%. The observedreduction in third instar mortality in the releaseplot may be atfibutable to an application of oxamyl on 16 October (Table 1) againstcotton fleahopper. Summyet aL (1992) reported 100% mortality of C. grandis one hour after application of oxamyl on potted cotton plants and observeda residual eflect of mortality for 16 to 20 days. In our study, parasitoid activity may have been impactedduring three successivecohort dates;5, 12, and 15 October.The 12 Octobercohort date registered the lowestparasitism (<20o/o) and coincidedwith the period of longestexposure by parasitoidsto oxamyl residues. The 5 and 15 October cohort samplesregistered parasitism of 63 and 50olo,respectively, apparently because the exposure dates of these eohorts representedshorter times of exposureby parasitoidsto oxamyl residues,3 and 13 days, respectively. Regardless,the impact observedas a result of the oxamyl applicationwas less drasticthan that observedby Summyet al. (1992). The high initial mortality (> 70%) recordeddwing the early infestation period in the C. grandis plot apparently resulted in a slower population increase and lower overall densitiesof boll weevil and percentdamaged fruit (Fig. 2), which comparedfavorably with densitiesand damagerecorded in the check plot with I I insecticideapplications for boll weevil (Table l). This corroboratesother studieswhich showedthat the greatestimpact of C. grandis releasesoccurs during the first two boll weevil generations(King et a1.1993, Colemanet al. 1996).In addition,the insecticideused to control suckinginsects is also employedfor control of A. grandis and probably affectedparasitoid survival. On the other hand, it is interesting that secondarypest resurgencewas not observedat this locatiorl perhapsbecause pesticide applications were not generalizeduntil Octoberor becauseofthe broadspectrum ofinsecticide used (Table l). In both locations, results showedthat augmentativereleases of C. grandis can inflict significant mortality on boll weevil populations,primarily due to parasitismof third instarso 1l but also of secondinstars and pupaeeither by parasitismor from feeding or venomization by adult C. grandis females(Vargas-Camplis 1998). When sustainedhigh mortality of first and second-generationsofbol wlevil is obtainedas a consequenceof C. grandisreleases, effective suipression of boll weevil populations may be feasible in t1re absence of insee+ieide"ppti"utio*. Howeven lour parasitisrnrates of irnrnaturesduring eady season will likely iesult in higher late-seasonpopulations of weevils that require repeated insecticidi applicationsto reducepest damage.In order to maximize suppressivepotential of biocontro-l-agentssuch as C. gandis. it is exbemely important to carefirlly integrate chemical control measuresfor boi weevil or other insect peststo avoid deleteriouseffects such as direct mortality of release agents and extant beneficial fauna that regulate secondarypest populdtions. ACKNOWLEDGMENT we greatly appreciatethe economic support provided by the USDA-FAS througf Grant No. fC-lO<-tOO, Project No. IvD(-ARS-I. We also acknowledgethe technicall supportprovided by Enrique GarzaUrbina and Joel Avila Valdez' LITERATURE CITED coleman,R. J., J. A. Morales-Ramos,E. G. King, and L. E. Wood. 1996.Suppressionof the boll weevil in organic cotton by releaseof Catolaccusgrandis as part of tho Southern Rolling Plains Boll Weevil Eradication Program, pp. 1094. In: Proc- Beltwide Cotton Conferences1996, Vol. 2. National Cotton Council of America' Memphis,TN. of King,-boll E. d., f. R. Summy,J. A. Morales-Ramos,and R. J. Colemao.1993. Integration weevil biological control by inoculative/augmentativereleases of the parasite Catolaccus grondit in short seasoncotton, pp. 910-914. 1n: Addendum to 1993 proceedings Beltwide Cotton Conferences.National Cotton Council of America. MemphisTN. Scott' 19951 King,- E. G., R. Col"-un, L. Wood, L. Wendel,S' Greenberg,and A. W' Suppressionof the boll weevil in commercial cotton by augmentativereleases of a waspparasite, catolaccus grandis,pp.26-30.12: Addendum to Proc.Beltwide cotton Conferences1995, National Cotton Council of America,Memphis' TN' Krebs, C. J. 1985. Ecology: the experimentalanalysis of distribution and abundance' Harper& Row Pub.,New York. 800PP' Morales-i{amos,J. A., K. R. Summy,and E. G. King. 1995. Estimatingparasitism b} Catolaccusgrandis (Hymenoptera:Pteromalidae)after inundative releasesagainst the boll weevil (Coleoptera:Curculionidae).Environ. Entomol. 24: l7 18'1725' Southwood,T. R. E. 1978.Ecological methods. Chapman and Hall, New York. 524pp. Summy,K. R., J. A.. Morales-Ramos,E. G' King, S. M. Greenberg,and A' W' Scott,Jr tbgZ. nietO evaluationsof the boll weevil parasiteCatolaccus grandis in the lower Rio GrandeValley, pp. 4l-50. In: 1992-1993Research report, Rio Farms,Inc. Monte Alto, Texas. Sturm, M.M., and W.L. Sterling. 1986.Boll weevil mortality factorswithin flower buds of cotton.Bull. Entomol.Soc. Am. 32:239'247. Vargas-Camplis,J., R. J. Coleman,J. Gorvalez,and L. A. Rodriguez-del-Bosque.1998' Outcome of two-year study of boll weevil confol with inundative releases of Catolaccusgrandis (Hymenoptera:Pteromalidae) in Tamaulipas,Mexico, pp' 1292- 1296. In; Pioc. Beltrvide Cotton Prod. Res. Conferences1998, National Cotton Councilof America,MemPhis, TN. 75 vol.29 NO.1 SOUTHWESTERNENTOMOLOGIST MAR.2004 SCIENTIFICNOTE THELOHANIASOLENOPSAE (MICROSPORIDLA) INFECTION IN ^tOrArOPSl,S INWCTAI CORRELATED WITH INCREASED ARTHROPOD DIVERSITY Matthew S. Brain, Jerry L. Cook, and TamaraJ. CooP Departnrentof BiologicalSciences, Sam Houston State University, Huntsville,'fX77341 Solenopsis invicta Btnen (Hymenoptera: Formicidae) has invaded most of the southem United States and Puerto Rico, decimating endemic ant communities and disrupting native arthropodpopulations, impacting biodiversity at many levels (Porter and Savignano1990, Gotelli and Arnett 2000). Cook (2003) demonstratedthat selective managementof S. invicta could lead to significantly higher endemicant diversity. Infection with the microsporidianpathogen, Thelohania solenopsae Knell, Allen, and Hazard, leads to smaller mounds, decreasedcolony density, lower queen weight and fecundity and decreasedsurvivorship of S. invicta (Briano et al. 1995, Briano and Williams 1997, Williams et al. 1999,Cook2002, Oi andWilliams 2002,Cook et al. 2003). Our objective was to determine whether endemic ant and other ground dwelling arthropod diversity is higher in areaswhere.S. invicnis infectedwith T. solenopsae. Between January and March, 2001 we conducted surveys for the pathogen I solenopsae(see Cook 2002 for sampling protocol) at the Sam Houston State University Center for Biological Field Studies (CBFS), 5 krn northeastof Huntsville, Walker Co., Texas,and Camp Swift (CS), an ArmyNational GuardTraining Site 13 lcrn S of Elgin, Basfrop Co., Texas. Deep sandy soils, mixed grasses,and loblolly pine forestswith interspersedhardwoods, with infrequentvehicle and foot traffic, characterizedboth survey areas. The resultsof this survey, and of prior surveys(Cook 2002), indicatedthat although T. solenopsaeinfection was geographicallywidespread, locally it occurred in relatively small infectionpockets. Therefore,to assessthe effect of infectionon local diversitywe restricted this study to one small arca at each suwey location where prevalenceof Z solenopsaewas greaterthan70 o/o. At each study site, two 6x30m plots were established:one in an area with Z solenopsaeinfection and a controlplot of the samedimensions in an areawith similar soil t1pes,vegetation and mound densities,but free from infejction. The number of moundsin each plot was recorded and mound volumes were derived using the formula of a hemispheroid(Porter et al. 1992). Ant and arthropod communitieswere sampledusing eight pitfall traps per plot, installed the week of 9 April 2001, arrangedlinearly four meters apartand collectedafter sevendays. Ants were identified to species,other arthropodswere identified to order, and a Shannon'sdiversity index was calculatedfor eachplot. Mormd volume comparisont-tests were performedusing SigmaStat (1997) software. Eight of l0 moundsin the CBFS infectedplot were infected w..rthT. solenopsae,and mound densityin the infectedand uninfectedplots was 0.055 and 0.044 moundsper rnl; respectively. Five of seven mounds in the CS infected plot were infected with ln lHymenoptera:Formicidae ' Correspondingauthor, e-mail: [email protected] 7' 7 solenopsae,and mounddensity in the infectedand uninfectedplots was 0.039 and 0.033 moundsper m'; respectively.Although mound density was higher in both infectedplots, averagemound volume in the CBFS infected plot was significantly smaller than in the uninfectedptot (1,341 cmt vs. 1,767 cm3;-t= 3.2, df = 16, P = 0.05). Averagemound volume in the CS infectedplot (1,523cm') also was smallerthan in the uninfectedplot (1,748cm3) but the differencewas not statisticallysignificant (t = 7.42,df - I l, P = 0.172). Similar numbersof S. invicta individualswere collectedfrom both infectedand uninfectedCBFS plots (Table l), but the infectedplot containedtwice as many nativeant speciesand almost five times more native ant individuals (Table l). Ant community diversitywas higherin the infectedplot (H'=0.428)than in the uninfectedplot (H'=0.172). The same arthropod.groups were collected in both plots; however,the infected plot contained30% more non-ant arthropodindividuals (Table l). Arthropod communify diversity,excluding ants, was higherin the infectedplot (H'=0.402)than in the uninfected plot (H'=0.357). TABLE l. Numbersof Ant Speciesand ArthropodOrders Collected in Two Field Plots Ants/ArthropodsCollected Uninfected Infected Uninfected lnfected So I eno p si s i nvi ct a Burerr 183 187 401 t?u D orymyrmexins anus (Buckley) t4 50 Par atr e c hin a vividula (Nylander) 33 5l Br achyrnyrmex d ep i lis Emay 12 Forelius mccooki(McCook) l- Forelius pruinosl's(Roger) t:t t25 Monomoriumminimum (Buckley) : l6 Total ants 283 552 518 Total non-^Linvicta ants 101 l5l l9l otlOther Athropodsrpoc Collembola 939 620 837 Arachnida 48 54 t72 Isopoda l3 4 22 Thysanura 15 10 l6 Lepidoptera(larvae) 13 I Coleoptera 9 J 1l Diptera 11 9 4 Homoptera 2 15 Hymenoptera(non-ant) 2 : 7 Onhoptera I I 2 Phasmida I Total non-ant 1053 7t0 1087 "Centerfor BiologicalField Studies,Walker Co., Texas o CampSwift, BastropCo., Texas Similar impacts to terrestrial ant domination were seen at Camp Swift. Only one nativeant species,Forelius pruinosis (Roger),was collectedin the uninfectedplot, while three native specieswere collectedin the infectedplot (Table l). A more diverseant communitywas presentin the infectedfield plot (H'=0.421) than in the uninfectedplot (H':0.255). Similar arthropodgroups were collectedin both infectedand uninfectedCS plots (Table l), but the diversity of the infectedplot was greater(H':0.502) than in the 78 uninfectedplot (H'= 0.43). The infectedplot contained53.1olo more non-antarthropods than the uninfectedplot. The fact thai similar numbersof frre ant individuals were collected in both infected and uninfected plots, yet infected plots supported more diverse ant and arthropod communities suggests that ?. solenopsae infection impacts the health and foraging efficiencyof individual fire ants,and consequentlytheir ability to dominateresources and displacecompetitors. Direct impact of the infection on queenfecundity probably accounts for most of the mound volume differencesfound in this survey, as brood production and worker numbers decline throughoutthe course of infection (Oi and Williams 2002). Decreasedforaging intensity, however, may secondarilyimpact colony health as the levelof collectedfood decreases,possibly applyng furthernuhitional stress to infectedqueens and developingbrood. ACKNOWLEDGEMENT Funding provided by Texas Army National Guard Environmental Resources ManagementBranch, LITERATURECITED Briano,J. A., R. S. Patterson,and H. A. Cordo. 1995.Relationship between colony size of Solenopsis richteri (Hymenoptera: Formicidae) and infection v/rth Thelohania solenopsae(Microsporidia: Thelohaniidae)in Argentina. J. Econ. Entomol. 88: 1233-t237. Briano, J. A., and D. F. Williams. 1997. EIfect of the Microsporidium Thelohania solenopsae (Microsporida: Thelohaniidae) on the longevity and survival of Solenopsisrichteri (Hymenoptera:Formicidae) in the laboratory. Fla. Entomol. 80: 366-372 Coolq J. L. 2003. Consewationof biodiversity in an areaimpacted by the red imported fire ant, Solenopsisinvicta (Hymenoptera:Formicidae). Biodiversity Conserv. 12: 187- 195. Cook, T. J. 2002. Thelohaniasolenopsae (Microsporida: Thelohaniidae)impact on red imported fire ants, Solenopsis invicta (Hymenoptera: Formicidae) in natural, unmanagedsystems. Environ. Entomol, 3l: l09l-1096. Cook, T. J., lowery, M. 8., Frey, T. N., Rowe,K. E., and Lynch L. R. 2003. Effect of Thelohaniasolenopsae (Microsporida: Thelohaniidae)on weight and reproductive statusof female alates of the red imported fire ant, Solenopsisinvicta. J. Invert. Pathol.82: 201-203. Gotelli, N., and A. Amette. 2000. Biogeographiceffects'of red fire ant invasion.Ecol. Letters3: 257-261 Oi, D. H., and D. F. Williams 2002. Impact of Thelohaniasolmopsae (Microsporidia: Thelohaniidae) on polygyne colonies of red imported fire ants (Hymenoptera: Formicidae).J. Econ.Entomol.95: 558-562, Porter,S. D., and D. A. Savignano.1990. Invasion of polygynefire ants decimatesnative antsand disrupts arthropod community, Ecology. 7l:2095-2106. Porter,S. D., H. G. Fowler,and W. P. Mackay.1992.Fire ant mound densities in theUnited Statesand Brazil (Hymenoptera:Formicidae). J. Econ.Entomol.88: 1233-1237. SigmaStat Statistical Software, 1997. Users manual, version 2.0 SPSSInc., Chicago,IL. Williams, D. F., D. H. Oi, and G. J. Knue. 1999. Infection of red imported fire ant (Hymenoptera: Formicidae) colonies with the entomopathogenThelohania solenopsae(Microsporidia: Thelohaniidae). Biol. Micro. Control92: 830-836. 79 vol.29 NO2 SOUTHWESTERNENTOMOLOGIST JUN.2004 COMPARISON OF ABSOLUTE ESTIMATES OF THRIPSTABACI (TIIYSAI.IOPTERA: TI{RIPIDAE) VTIII FIELD VISUAL COLINTING A}.ID STICKYTRAPS INOMONFIELD IN SOUTH TE)(AS Tong-Xan Liut and Chang-ChiChu2 lVegaable IPM Iaboratory, TexasAgriculhual ExperimentStation, Toras A&M Univemity,2415E.Highrvay 83, Weslaco,TX 285968399 'USDA-ARS,^ West€rnCofron Research Labordory, 4135 E. Broadwan Phoenix,AZ 85040 ABSTRACT Absolute estimalesof onion thrips, r?rripr t6aci Lindemlur on onions were used to determinetbe reliability of field visual cotmting and blue and rvhite plastic cup haps and cc trapa for monitoring thrips in onion fields. It took >140 min to sampleone plant for the absoluteestimates of thrips, which was =l5-fold longer thnn neededto sampli one plant by field visal counting ad 3.8- and 4.3-fold longsr thm a -.mFle using a plastic cup trry or a cc rap surple, respeotively.Results indicated ttrat aaun thrips compriseds16.4 and 15.87oof total thrips in the absoluteestimates and field vizual comaing respectively, and were well conelated with total thrips in each sampling netbod (r:0.81 and 0.73, rcspectively).Toal tbrips and adults by field visual coqnting estimated45o/o of total ttuips and rfE% of adults of ttre absoluteestirnates, and were highly correlated with the absolute estimates(r = 0.98 and 0.95, respectively). Blue plastic crry traps caughttbe most 6rips (19-23 thrips/tra/day), followedby white ptastic cup rraps (lGl2 thipsnrap/day), comparedwith INTRODUCTION Oniot (Allitan cepaL.) is a qajor vegetablecrop in south Tq 83 Sticky traps ofdifferent colors, materials,and shapeshave been used for sampling and monitoring, estimating populations, and controlling various species of thrips, including T. tabaci, Tlrips palmi (Kanry), urd, FranHiniella occidentalis @ergand€) wrder greenhouseand field conditions (Lu 1990, Cho et al. 1995, Tsnchiya et al. 1995, Vernon et al. 1995,Terry 1997, Roditakis et al. 2001, Szenasiet al. 2001). The CC trap was initially developed for monitoring the activity of silvedeaf whitefly, Bemisia ugenifolii Bellows & Perring, in cotton and other field crops (Chu and Hemeberry 1998,Chu et al. 2000). Besidesbeing usedfor trapping8. wgentifolii, modified CC traps have beenused for tapping various other insects,including leaflroppers,Empoasca spp., andF. occidenfalrs(Chu et d. 2000). Thesemodifications for CC traps include chmStnS colors of the trq base(yellow, rum, r€d, line geen, spring green,woodland gl€€'n,tnre blue, white, and black). The objectivesofthis study were to determinervtether sticky traps can be used for monitoring thrips population dynamicswith comparisonrvith absoluteestimase and field visual counting underfield conditionsin sodh Texas. MATERIALS AND METHODS The study was conductedat the Reseach Farm of the Tq 84 Products Inc., Chelmsford, MA) thd admits light for afult orientation to the trap, a deflector plate that pr€vcnts insects from escapingfrom the trap, and a.blue or white cylinder bas€ with an open-endedcontainer attenuded cone tbat allows adult entmce (Chu et al. 198, 2000). The CC trap used in this study was modified by rerroving the deflector plate and coating the inner surfaceofthe clear cup with sticky Tanglefootglue (Iangle-Tr4 Insect Trap Coding, Aerosol formulg The Tanglefoot Compann ffid Rapids, MI). The plastic cup trrys w€re coated with Tanglefoot glue on the outside surfrce only. Both the plastic cup traps and CC traps were individually hung on a wooden stakewith an iron wire hook 2-3 w abonetbe plant canopy.The height of the traps ums adjustd wi0r growth of the plants. In eachplot,20 plastic cup traps (10 of each color) and 20 CC traps (10 of eachcolor) were randomlyplaced in the field at a distanceof =3-5 m fiom the nearesttrap. The taps were placed in the field in the eady noming. After rcmaining in the field for 24bo they were brought to the laboratory,and all thrips on the tap6 w€re id€ntified and cormted.Traps were placedin the field weeHy on the sameday as the nhole onion plants urcre smpled. The time usedto samplethrips on sticky tr4s, including labeling, assembling,painting with Tanglefootglue, installing traps in the field, co[ectittg and returning traps back to the laboratory and counting thrips was recordedon 14 February. Numbersof thrips collected from onion plants and sticky taps, and the time used for sampling thrips on onion plants or traps rrere analyzedusing analysis of variance (SAS Institute 2002). Numbersof adult thrips on CC traps were poolcd for data analysis becauseno significant differenceswere formd betweentrap colors. Meenswer€ separated using the honestysipificant ditrer€nc€test or Tukey t€st aft€r a significant F-test at p = 0-05 (7n 1999). Becauseonly a few prpae ( RESI.'LTSAI{D DISCUSSION Only T. taboci wu formd in the experimentalfield in 2000. Thips w€rc pres€m on onion plants from early Februaryrmtil harvest with peal6 in early March and early Aptil (Fig. l). Thrips densities were high throughout the seasonrelative to those in previons yean (spaks st d. 1998, LirL unpublisheddata). An averageof 221.6* ll.l thrips were found on eachonion plant in the absoluteestimat€s, of ufrich 34.g + 3.7 were adults or 15.7o/oof lclal thrips. An averageof 166.8+ 21.8 thrips was cormtedby field visual courmtingof rryhic'h26.4 + 3.1 rver€adult 6rips ot 15.8o/o.Field visual cormtingsof Fqloq thrips and adults thrips urcnesignificantly t6s rhan the abaoluteestimateslF= 13.91;df=1,9;P=0.0O57forallthrips;andF= 10.74;df = 1,9;p=0.0096foradult thrips) (Figs. l, 3). Field visual counting accormtedfor z5.l afr78.o/oof total rhrips and 4* tb"ipo of the absolut€estimates, rrespectively. Of the total thrips ftom onion ptants, 84.3 md 84,1o/owerelarvae (including pupae),and 15.7 and l5.9zo urcreadults fiom the aholute €stimatcsand field visual counting respectively. _Significantlymore adult thrips wenebaped on the plastic cup faps than on cc traps(F= 67.74-73.34;df = ,261; P4.001) (Figs.2, 3). Of the two colorsof plasticcup traps, blue traps caught significantly more tbrips tlun the white trqs (F - 11.12,tr = i, 283;?{.001). How€ver,there wpre no sigrificant differericesin numbersof adult thrips caughton thetwo colorsof the CC traps(F= 0.66;df = l, 261;p - 0.416g)(Fig. 3). Numb€rs of total thrips and adult tbrips in the absolute €stimateswer,e well conelatedwift total tluips and adult thrips by field visual counting (r = 0.98 and 0.95, p < 0.0001, (Table 1). Meanwhile, numbers of sddtthrips rve'e relatively 85 well conelated with total thrips in both the absoluteestimates and field visual counting with r-values at 0.81 and 0.73, respectively.Fwthennore, numbers of adults ttuipa in field visual counting were well correlatedwith that in the absoluteestimat$ (r = 0.95; P < 0.0001). + Absoluteestlmate! +- Fleldvbual countlng -+ Adulb in ab3olut€€stimate! *F Adults in tleld vbual IIJ o n 300 c -g bo .F zoo E F 100 2nto0 2l21loo 3/600 3,norco 4t3too 4l17too 5t1lo0 Date (m/d/y) FIG. l. Absolute estimatesand field visual cormting of Thrips tabaci ot onion plants (Spring 2000, Weslaco,Toras). TABLE l. Correlationsamong the Mean Numbers of T. tabrci on Onion Plants and MeanNr.mbersof Adult Ttuips on Traps(Spnng 2000, Weslaco,TX) Correlationcoefficients, r oounting: estimates: counting: cllp cup frp all thrips adults adults taP taP : all thrips Field visual 0.83b 0.73' 0.23 0.18 0.61 counting:all th'rips b Absolute estimates: 0.95 0.07 0.38 0.45 adults Field visual 0.09 0.38 0.45 cormting:adults Blue cup trap 0.87b 0.08 White at P = 0.05 , respectively Numbersof adult tbrips caught on the blue plastic cup taps were well conelated with those on the white plastic ctry 6qs (r = 0.87; P = 0.001l), but the're were no = signifioant correlatio$ betweenthe plastic cup trps and the CC haps (r 0.08-0.05;P > 86 0.05) Clable l). There were also no sipifcant cor€lations in numben of tbrips counted on onion plants and the adult thrips caughton both the plastic cup baps and the CC taps (r=0.M-0.61;P>0.05). + Blue cup trap ur 80 + Whlt€ cup lrap U' "'t- CC trap .t{ -v- Ablolut€ €atlmate3 c + Fleldvbual countlng .E cL 60 o CL E g40 c t E20 2ntoo 2t21t00 3/6/00 3t20too 4t3too 4t17too Date(m/dly) FIG. 2. Tlrips tabaci adults countedon onion plants and caught on traps in ihe onion field (Spring 2(XX),Weslaco, Toos). A" 0n plants rAE llt ,r.t 2ffi caw FVC v, $ .t{ 20 *l o. E 150 g o. Ers tt .g 1oo g "c 910 F t All thrip Adutlg Cuptrap CCtrap FIG. 3. Ov€rall numbersof T. tabaci on onion plants and adults caughton sticky haps (spring 20(D, weslaco, Texas). The same letters over the paired bars indicate that the meansane not significmtly diffq,ent d P: 0.05 (Tukey Test, SAS Institute 2002).AE - absoluteestimates; FVC - field visual corurting. 87 oUJ 'tto ff e E 'D .E 100 F FVC CupTnp CC T.rp FIG. 4. Comparisonof time (in ninutes) usedfor samplingT. tabaci o^ onion plalrtsand sticky traps ftom onion field (Spring 20fi), Weslaco,Texas). The sameletters over all bars indicate that the meansare not signfficmtly different at P = 0.05 (Tukey Test, SAS Institut€ 2W2). AE - absoluteestimates; FVC - field visual cowting. Time used for sarrpling tbrrips on onion plants and tb€ sticky traps was remrkably different (F = 45.27; df = 3, 59; P 4.0001) (Frg. 4). It took >140 minutes/personto processa single absolutesample Glan$ from prepatrion to counting all thdps on each planl urhich was almost a l5-fold increase in time oomparedto cormting all thrips on a plant in the fiel4 and a 3.8- and 4.3-fold increaseof time for processinga plastic cup tsap sample and a CC tap sample,respectively. The absolute estimatesare the most time costly; however,the estimatesare of utmost importancewhen the reliability of other relative estimatemethods is evaluated.The field vi$al counting method, either counting all thrips or only adult tbrips on onion plants, was the besg providing relatively reliable estimatesof field thrips populationwith lesstime. Trapping tbrips with mlored traps has beena generalpractice for monitoring and samplingthrips although the taps measuredactively flying adults in the field while the whole-plant counts included all thrips on the onion plants (Lu 1990, Terry 1997). Generally, blue and vhite have been conside,redas the preferredor the most preferred colors for several speciesofthrips, including T. tabsci. Although the blue taps caught sigrificantly more thrips than the white onesin this study,which was consistentwith the prwious findingF (Lu 1990,Cho et al. 195, Terry 1997,Chu et d.2000), we found that both the plastic cup traps and the CC traps w€re not usefirl for monito'ringand sampling thrips rmderfield conditionsin south Texas. ACKNOWLEDGMENT We thank R. McGee (Texas furiculture Experiment Station, Weslaco)and J. A. Bayer (USDA-ARS, WesternCotton Researchlaborafiory, Phoenix, AZ) fot reviewing the early draft of this manuscript,and J. Martinez and M. Moral for technical assistance. 88 Publication of this manuscript has been approved by the Director of the Texas Agricultural Experiment Station at Weslaco, and the Head of the Department of Entomology,Texas A&M University, CollegeStation, Texas. REFERENCESCITED Anonymous.200l.2000 TexasAgricultural Statistics,Texas Deparhent of Agricuhre Bulletin 258, TexasAgricultural StatisticsSenrice, Austin, TX. Cho, K. J., C. S. Eckel, J. F. Walgenbach,and G. G. Kennedy.1995. Comparison of colored sticky traps for monitoring thrips populaions (Thysanoptera:Tbripidae) in stakedtomato fields. J. Econ. Entomol. 30: l7Gl90. Chu, C. C., and T. J. Henneberry.1998. Developmentof a new whitefly fiap. J. Cotton Sci.2: 104-109. Chn, C. C., P. J. Pinter, Jr., T. J. Henneberry,K. Umed4 E. T. Natwicb Y. A. Wei, V. R Reddy,and M. Sbrepatis.2000. Use of CC frapswith different trap basecolors for silverleaf whiteflies (Homoptera:Aleyrodidae), thrips (Thysanoptera:Thripidae), and leaflroppers(Homoptera: Cicadellidae). J. Econ. Entomol. 93:.1329-1337. Edelsoq J. V. 1985. A sampling method for estimating absolutenumbers of thrips on onions.Southwest. Entomol. l0: 103-105. Edelson. J. V., B. Cartudght, and T. Royer. 1986. Disfiibution and impact of Tlnips raDaci(Thysmoptera: Thripidae) on onion J. Econ Entomol. 79:-502-505. Lu" F. M. 1990. Color preferenceand using silver mulchesto conhol the onion thrips, Tlrips tabaciLindeman.Chinese J. Entomol. l0:. 337-342. Royer, T. A., J. V. Edelson, and B. Cartwdght. 1986. Damage and contol of Thrips tabaci Lindentr on spring onions.J. Rio GrandeValley Hort. Soc.39:.69-74. Roditakis, N. E., E. P. Lykouressis,and N. G. Golfinopoulou. 2001. Color prefercnce, sticky trap calches and distribution of westem flower thrips in greenhouse cucumber,sweet pepper and eggplantcrops. Southwest. Entomol. 26: 227-237. SAS Instia$e. 2002. SAS/STAT users' guide,Version 8.01, Cary, NC. Shir€lCF. H. l%8. Collecting and cormtingonion thrips in the Winter Gardenin 1953.J. Fron. Entomol. 47: 616.618. Sparks,A. N. Jr., J. Anciso, D. J. Riley, and C. Chambers.1998. Insecticidal conhol of thrips on onions in south Texas: Insecticide selection and application methodology.Subtrop. Plant Sci. 50:58-62. Szenasi,A., G. Jenser,61 1.7mn.2001. Investigationon the colour geferanceof Thrips tabaci Lrndeman (Thysanoptera: Thripidae). Acta Phytopathol. Entomol. Hungarica36: 207-211. Terry, L. l. 1997.Host selection,communication and reproductivebehavior, pp. 65-118. In T. Lewis [ed]. Ttrips as crop pests.CAB International,Wallingford, UK. Tsuchiy4 M., S. Masui, and N. Kuboyama. 1995. Color aftraction of western flower thips (FranHiniella occidentalisPergrande. Japanese J. Appl. Entonol. 2.oo1.39: 313-319. Vernon, RS., and D. R. Gillespie. 1995. Iinfluenceof nap shape,size, and background color on captures of Franfliniella occidentalis (Ihysanoptera: Thripidae) in a cucumbergreenhouse. J. Econ. Entomol. 88: 288-293. 78,J.H.199. Biosatistical analysis,46 Edition. Prentic.e-Hall,Englewood Cliffs, NJ. 89 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JL,N.2004 GUT CONTENT ANALYSIS OF TIIE SPIDERH/BI.MI INCURSA (ARANAE:AITIY?HAENIDAE) USING SEROLOGICAL METHODS J. J. Re,nouard,R. Creamer,D. B. Richman Departnent of Entomology,Plant Pathology,and Weed Scie'lrce, New Mexico StateUniversity, Las Cruces'NM 88003 ABSTRACT We report the developmentof a techniquefor analping the gut cont€ntsof spidersusing serological methods. We used pecan aphids, pests of economic importance in the southwest,urd the spiderHibana inanrsa(Chmb€rlin), conmon to pecanorchads. Two dimensional SDS-PAGE was used to isolate proteins of blackmarginedpecm aphid that were distinct from the spider. These proteins were used to manufacture polyclonal antibodies that oould detect ryhid proteins. Cross-absorptionwas used to increasethe specificity of these antibodies. We used indirect ELISA to show that proteins originating from blackmarginedpecan ryhid could be detectedin the gut of H. inqrsa after feedingon the aphids in t hboratory e'nvironmerit. Starved spiderstested negative. H. inc'ursa urd other spiders collected from aphid-infested pecan groves also tested positive for the pr€s€nceof aphid proteins. INTRODUCTION Development of laboratory methods for studying arthropod predatorsof crop pests is neededto bett€r implemeirt integratedpest control. Hunting spidersare dilficult to study becausethey predigestfood and do not constructwebs, leaving little evide'lrceof their prey. However, the relationship between such predators and pests must be well undetstoodif changesin pesticideuse are to benefit agriculturalists. The objective ofthis researchwas to develop an immunological technique to determine if a particular arthropod predator had consumed a particular prey species. This research focused on a member of the Anyphaenidaefamily of spiders (Plafirick 1974),Hibana incarsa (Chamb€rlin) @rescovit 1991). This speciesis a foraging (non web building) spiderprevalent in pecanorchards in southern Nerv Morico. The prey speciesexrnined was the blackmrgined pecan 4hid Monellia caryella (Fitch). Pecm 4hids are c4able of inflicting a grerit deal of damageto pecan t6 lcnrya illinoinensis (Wangenh)K. Koch]. Aphids drain a tree's €Nrergyres€rves, which has a direct impact on fiuit production (Teddersand Wood 1985,Wood et al. 1987)' Many varietiesof pecansare alt€matebearing cultivars, and danage during nonbearingyears will alfect the crop load the following year as well. Biological control agentsare an important factor for managingpecan aphids (LaRock and Ellinglon 1996, Liao et al. 1985), 6d spiders rc increasingly recopized as a vital componentof the biocontsol agent complex @uruoongsook et al. 1992, Richman 2003' Riechert and lockley 1984). Hibana gracilis (Hentz) was observed feeding on blachnargined aphidsin the laboratory@umroongsook etal. 1992). In addition, IL inanrsa was obseryed feeding on both blackmargined pecan aphids and black pecan aphids, 9l Melanocallis caryaefoliae(Davis) in the laboratory and, after eating,the abdomensof thesE take on yellow ltfders or.black discoloratio4 respecriviy Ri"hmin t003). This suggests that thesespiders are willing to prly on pec- upirids,".ti itr"t cespit- trte;re-digestiin of 14el nre.v,-some portion of it will remain in rhe gur, so that ii could be deiected by biocheuricalmeans. - Serological and molecular anallaes have been used to monitsr arttuopod predation (Greenstone 1996, sunderland 1996). ELISA was used to detect remains of aphids in various insect predators(Sunderland et al. 1987) and serological studieswith monoclolral antibodies have been usgd on spider-aphidsysterns 6Harwooa et al. 2001). polymerase chain reaction @CR) hasbeen reported for detectionoiaphids in insects(Chen et ai. ZOOO;, a mite (cuthbefison et d. 2003), and spiderpredators (Greenstone and snuaan 2003). Tb; PCR detectionof a leafiropper,Nilapamata lugens(StLl) in its spiderpredator has also been reported (Lim and Lee 1999).. The method presentedhere uses an ELISA assay with polyclonal antibodies,a faster, less expensivesystqn. we slrow that a polyclonal iLIsl, systernis specific enoughto distinguishprey proteinsfrom thoseofa predatoi. MATERIALS AND METHODS Aphids and spiders were collected from pecan orchards in Las cruces, NM. Both blachnargined pecanaphid and black pecanaphid specieswere collectedby aspirationtaps from tees,,either directly, or in canvasbags or sheetsfollowing beatingbf [aves. Thiy were stored in plastic vials, containing one or two pecan leaves. Aphids were used for feeding experimentswithin three days after collection. Unused4hid sarrpleswere froze,r for use at a later date.Hibana inctrsa (chamberlin), Theridion sp., and philodromtu sp. spiders were collected from hees by beating tree branches. Spiders were captured from beating sheets or bags, by hand, in small plastic vials. spiders were either frozen immediately or used for feeding experiments. (Jloborus glomosus (walckenaer) (Llloboridae) and the insectstested, Myzus persicae (Sulzer), Diabrotica undecimpurrctata (Mannerheim)Hippodamia convergens(Guerin-Meneville), and chilocorus stignata (Say), were collected from greenhousesat New Mexico State University. All spiders used for. these experimentswere identified by DBR. Voucher specimenof spidersand aphidswere retainedin the NMSU Artlropod Museum. All spidersused in feeding experime,ntswere starvedfor a minimum of 24h to clear the gut of extraneousorganic material andprovided only water. Spidersof approximatelyequal carapncesize were selected for experimentsand contained in cylindrical plastic "Fed" vihs. spiders were provided with three aphids of the speciesbeing examined as well as watsr. Vials containing spidos were storedat room ternperature.Spiders were observedat 24,48, and72 h to determineif feeding occurred,visualized by the absenceof one or more .p4&: when feeding occured, spiderswere frozen and stored at -20oc for later protein analpis. Samplesof either one spider, fed or starved,or three aphids of the samespecies, were preparedfor sDS-PAGE. samples were ground in 100 pl Laernmli buffer (0.125M Tris, pH6.8, 4o/ow/v SDS, 20Yovlv glycerol, l0%ov/v 2-mercarptoethanol)in microfuge tubes, using small pestles. Sampleswere then boiled for l0 min" and immediately loadedonto a preparedgel for electophoresis. The first dimension was performed in tube gels containing 8 M urea, l2yo acrylatride, 2% Nonidet P-40 non-ionic detergent,and 5Yo anpholytes in the pH 5-8 range. During this electro'phoresis,the upper chamber contained 0.02 M sodium hydroxide, and the lower chamber contained 0.085% phosphoric acid. The gel was prefocusedfor I h at 200v. Electrophoresiswas performedfor 16 h at 400v, followed by I h at 800V to tighten protein bands' Gels we,reremoved from the tubesand storedin equilibration buffer (0.2 MTis,2o/o SDS,20%glycerol, 0.01 M dithiothreitol)at -70"Cuntil used. 92 The seconddimension was performed using a Proteantr xi apparatus( Bio-Rad). Gels were composedof a l2Vo acrytamideresolving layer buff€red to pH 8.9, urd t7o/o stacking layer buffere.dto pH 6.7. Tube gels were laid acrossthe top of thesegels with the bottom of Ae tuUegel on the right hand si-rleof the seconddim€Nrsion gel, and coveredwith 250 pl of Laemmli-buffer. electrophoresisof this seconddimension was perfomredfor 16 h at l00V in nis-glycine buffer, pH 8.5. Twi dimsnsional (2D) SDS-PAGEgels were dweloped using silver stain (Merril et al. 1983). Stainedgels were scannedon a flatbed scarmerto pres€ntethe image. Protein spots used for antibody production were stained using an altemate method. Two dime'nsional SDS-PAGE geh were developedusing SYPRO Ruby stain @io-Rad) according to the product directions. Gels were removed from the apparatusand fixed in a solution of lE/o acetic acid/40%methanol for 30 min, with ge,lrtleagitation. The gel was rinsedbriefly with water, soakedin 175ml S\?RO Ruby solution for 2 to 3 h, and illuminated on a ultraviolet ligbt box while the protein spotsw€re excised. The 2D gel tecbniquesseparated proteins from aphids and spiders that fed on aphids, providing clear resolution of protein bands. Protein spotsuique to ryhids, but also found on gels ofspiders that had fed on aphids,were selectedfrom several gel comparisons,cut from fresh aphid gels stained with SYPRO Ruby, and used for antibody production. Pulverized gel fragments were injected subcutaneouslyinto New Taland White variety rabbits to gen€ratepolycloual antibodies. Booster shotswere given at two- and three-week interrrals. Blood was drawn at three-weekintervals and serawas removedby centrifugation. The IgG was purified from the serausing a Protein A sepharose(Sigma) column and diluted as necessaryto lmg/ml. Cross-absorptionwas perfomredto removenonspecific antibodies from the purified sera- To increasethe effectivenessof cross-absorption,ELISA was perfomted using dilferEnt ratios of spider and primary antibody. ELISA was performed as describedbelow. Spider massfor cross-absorptionwas varied as 0.5X lX nd2X of 21.4 mg p€r 900 pl. This mass was chosenbased on the averagemass ofan adultHibana. This averageincluded adultsof both sexes since rtL incarsa is not largely dimorphic and both sexes were used for experiments.Uobottts glomoms weighing 21.4 mg were frozen and pulverized in 900 pl ELISA antibody buffet describedelsewhere. Primary antibody was diluted to l:1,000; t:2,500; l:5,000; l:7,5@ and l:1O000. All tials comparedthe effective'nessof dilutions using aphi4 U. glomosw and aphid + U. glomosusmixture. Denaturationof spiderproteins by freezing and boiling for different time intervalswas also tested. Uobottts glomosus weighing 21.4 mg were frozen and pulverized in 900 pl ELISA antibody buffer or TBST (Tris-buffered saline, 0.5% Tween 20). To denature spider protein, this mixture was boiled for l0 min, frozen, boiled for 10 min again,and placed on ice. After cooling specific IgG fiom a rabbit was addedat a l:5,000 dilution. The cross- absorption mixture was incubated for 60 min at 37oC. The mixture was then microcentrifuged for l0 min at 14,000 RPM to remove solid debris and antibody aggregates.The mixture was usedimmediately for immunologicalpurposes. Indireot ELISA was performed to quantiff signal detection, as describedpreviously (Creamerand Falk 1989). Insect samples,primary antibody,and secondarygoat anti-rabbit conjugate were incubated for 2 h il 37oC. Primary antibody was diluted l:5,000 and secondaryantibody was diluted l:2,000, Plates were allowed to develop color for 15-60 min. Reactionswere determinedquantitatively by measuringabsorbance on a microplate reader.A difference in signal stength of at least 2.5X betweenEeatnerrts and contnolswas consideredpositive. 93 n6 ueced pau6rerrDtc?lqgo seldtnusq1,n fle,r4tsod peprar {poqnue peqJosq?-ssorceqJ ' Dilnaul'fi {nPeJo sseu u?9ru 'e eql uo PessqrDsoqc s?rn sssru slrlf, scolle lsoru s?^r rerBmqJo rl 996 red rap;ds tru ',no1 l'l7lo uoscquacuoc? 1eeJ$xru repldg sB,r\suoqe.qrbcuoc ueJeJJIp ssorce esuodser 'eARooIIe u e8ueqc eq1 qtnoqlp lsoru serr 000'g:I Jo uoFnIIp fpoqnw freruud y 'e^gceJJaeg ol acu€qro8q€-ssorc roJ uollnlos {poqnua Jo uonlpps eq1 o1 roud perqeuep fgtnoroql eq 01 psq tqgrosqe -ssorc roJ pesn semlxmr reprdg 'pe1ss1erel\ emlxnu reprds e,nlceer-ssorcpu? r$oqgm z(reuud go suoqe.qrocuocSurfrun 'EInseJlrelsrsuoc erour pecnpud eres peqrosqe-ssorc 'slpser tutsn qegq egqiu cpeue pecnpord ?Jes peqJosqe-sso.rrlnorpu\ spl4 VSITS 'eAnceIJe '('ds 'sraprds osp ere,$ uondrosqe-ssorcrog sn\capo,r|o-7)saopl^r {r"lg sts qcns raqgo 'sesnoquse.6 tursn slse; Fcol rnog pepefioc snsowolt 71Eursn peruro3ued se,u uoBdrosqe 'seldtues -ssorC ulelord Jo Jequmu ep1lr\? ol polcper-ssorc1eql solpoqrlus paurstuoc 'a,usuodser eldures umres snn 1eql papa^er Esel VSIIS 1soru eql eq o1 pelord gg urelord;o peelq pp1ul eql uro{ pagpnd sepoqquv 'sarpoqprr?puolc{1od acnpordol posn era,t qet FuorsueE-rpo,$l ulog pasp)€ prUdeuucad peudreuqcelq t|Io+ spwq ualord orrcurslp ered\ reqr surelord aresuods peroqumu r:t*5T'i'"gjr-#fi:iiTill'fr'T#1; -g {q pepredesspgde uecadpeqEreur4cBlq uo poJlerlt ssJncursu8qrHJo surelord 'I 'gIiI 'sassq48P tuqst:o ut suelord &re rlc1stu lorr p1p {eql ecus 'peuuuapp secuenbesplce ouun 'uopcnpord rcq1 tq,req o$dsap ?egurcpr lou e.nrn sureprd eq; ,(poqpue roJ rnsoqc erer$xnelord esorlJ 'ezls uI B(Ft gg {lapurrxordds ere,tl (l* pue g6) smelord prUdeoa41 'peguuept sunlord pgdu puqsrp moJ erelr araql '(1 'tr.g) spgde ueced peudrarrDtc?tq utog ftmuutlJo ss pognuepl erea\ sraprds peg uI spusq urelord 'sreplds pe JBls plr? peJ q1;mpqde uecadpeu€rerrDtc?lq uog sruelpd turpueq uplord eq1Suuedruoc fg NOISSNSSIC CI\IV SIlNS!ru pecana$!! aphid (Ams 0.83 +/- 0.35, n=7) md H.inanrsathat fed uponblackmarqlned antibodyreacted less strongly to /{' tO.gO+/- ii.tS, n=t2) in the laboratory. Cross-absorbed ir"urt o which hadbeen staned (0.L7+ l' 0.08,n=6)' of ELISA tests were afso performed on other insect speciesto determine the extent samples potential crossreaction $aUte t;. Cross-absortedsera reacted more shongly with of tht*.glo"d pecan aphid than with black pecan aphid and geen peach aphid. This sera also pt6Oo""d a *eako responseto cucumber beetle, Diabrotica undecimpunctata (Guerin), and Chilocortts stigmata ltvtannertriim;, or lady beetle,Hippodamia -is convergens were collected from iSuy). The'-cucumber beetle non-predacious; lady beetles greenhouseswhere no aphidswere observed. TABLE l. ELISA Reactionsof BlackmarginedPecan Aphid Comparedwittr Other Aphids and Beetles. Insect n (+S Monellia caryella(n4) 1.06+ 0.34 MeI ano c al lis caryaefo liae (n4) 0.65+ 0.20 Myzuspersicae (n=-2) 0.78+ 0.25 D iabr ot i ca und e cimpunc ta t a (n=2) 0.52+0.02 Hippodamia conv*gens (n=2) 0.23+ 0.01 Chi I oc orus sti gmata (t--2) 0.16r 0.04 Stawd Hibana inanrsa (n4) 0.38+ 0.25 The ELISA tests of spiders collected from aphid-infestedpecan groves gave sfrongly positive responses(Table 2). H. inanrsa andPhildromus sp. individuals reactedwith the antisera,while the Theridion sp. individuals did not. Of the two U. glomosuscollected from a greenhousewith aphid-infestedpecans, one showeda stong positive reaction,the other a negative reaction. Ditrering levels of reactivity within H. incursa was found, zuggesting that individuals with higher positive reactionsmight have fed on aphids more recently of consumedlarger numbersof aphidsthan spiderswith lower resporups. Thesetwo factors, amount of prey consumedand elapsedtime after feeding, have been shown to affect tho sensitivity of detection of pink bollworm eggs in predators using monoclonal ELISA (Haglerand Nara4jo 1997). TABLE 2. ELISA Reactionsof SpidersCollected from PecanTrees. Spiders Alos n. Hibana inatrsaQrl) 0.13 Hibana incarsa (n-3) 0.45 Hibana incarsa (n=l) 0.02 Theridion sp. (n=2) 0.01 Philodromussp. (n=1) 0.11 Uloborusglomosns (n=l) 0.39 Uloborusglomosns (n:l) 0.0r StawedHibana incursa (n=L) 0.01 The amountof time after which prey can be detectedin a spider appearsto vary greatly with the spider-prey combination, eveir when using the same qpe of detection method, monoclonalantibody ELISA. The leaftropper,Nilapamata lugens,could be detectedwithin the spiderPirata subpirafi'cus@dsenberg et Shand) for only six h after consumption(Lim and Lee 1999), while the aphid Sitobion avenae(Fabricius) could be detectedwithin the spider Lepthyphantestenuis (Blachvall) for more than 150 h (Harwood et al. 2001). 95 Additional researchwill need to establisha detectionthreshold for the quantity of aphids consumedper spiderand the time el4sed sinceconsumption ror o* syst"ii.-""[r"t h all the ELISA tests,- samples of fed spidgr and positive n'ria c spiders a shonger 1,19o""4 siryal than aphids alone. ihis result could have occurredbecause of, Ine mcrcased d€traturationo!3eta proteinsduring spider feeding compaed to the minimal denaturation during the ELISA sample preparation pnrcess. Since the antibodies were produced to denatrued {rroteins, ne priferential reactions with spider-denaturedaphid proteinsis not unexpected The polyclonal antibodiesy,sed for this procedurebind to multiple epitopeson the target which could potentially ryt:tt causecrois reactivity o. sensitivity rir"ui.-r. However, et (1987) jn luid€rla+ d' their work on predatorsor "*""t .pliis".p"l"a a detection limit of less than l/l00th of an adult aphid, and observedno cross reaction outside of Apryqid*. Using monoclonalantibodies, which target only a single epitopeon the proteirl would alleviate or eliminate cross-reaction,but are more cosfly and time consuming to produce and could have-lorrer sensitivity and shorter detection periods than polyclonal antisera (Sunderland 1996). Monoclonal antibodies have been used successfully in 9:lTtign of many predator-preyinteractions (synondson et al. 1999, i"glo -a Naranlo I997, GreenstoneI996). A recen! study demonstratedthat PCR could be usedto detectmitochondrial DNA from 3.pfy aphiq in a spider predator (Grwnstone and Shufran 2003). This techniqueis also highly sensitive an! wec!fic, but can only be performed oo pt"y ror wtricn genomic sequ€ncedata is availablefor the synthesisof specific DNA primer fragments. We have demonshateda simple, rapid and accuratemethod that caribe usedto detectthe gut cont€ntsof spiderpredaiors. Our ELISA basedtest is adaptableenough to be appliedto a variety of arthropodpredators and prey. Our methodselectsprey protei-ns ttrat are distinct from the predatorbeing studied. Polyclonal antibodiesto on" p*i"io limit the potential for cross-reactio4 but cannot eliminate it completely, as we observed. The eroneous antibodies must be removed, and cross-absorptionwas demonstratedto be effective for doing so. We also observedthat cross-absorbedsera showedless specificity for aphidsof species-other than the speciesused for antibody production. firis would Ue lmportant to pecan 19t-:.oh on aphids as there are severaldistinct speciesofpecan aphid which inflict differ€nt degreesof demage. Our methodhas beendernonstrated-to be elfiectivefor studies ofhunting spidersin the field, which pre-digesttheh prey, and do not leave signs ofwhat theypreviously consumedas web building spidersdo. ACKNOWLEDGMENT We thank Andrew Moya, JaimeRasco& ChristinaDragon, and JenniferRomero fot their assistancein_collecting and preparing insect sarnples. We also thank Carol potenza, and Jose ortega-curtruza forproviding technical advice. This work was supportedin part by the New Mexico StateUniversity Agricultural ExperimentStation. LITERATURECITED Brescovi! A. D. 1991. Hibana, novo g6nero de aranhas da familia Anyphaenidae (Arachnida"Araneae). Revta brasilia Entomol. 35: 729-744. Bumroongsook,s., M. K. Harris, and D. A. Dean. 1992. predation on blackmargined aphids(Homoptera: Aphididae) by spiderson p€crut. Biol. Coat. 2: l5-lg. chen, Y., K' L. Giles, M. E. Payton,and M. H. Greenstone.2000. Identifying key cereal aphidpredators by moleculargut analysis.Mol. Ecol.9:lgg7-lg9g. creamer, R, and B. w. Falk. 1989. charact€rization of a nonspecifically adphid- hansmitted cA-RPv isolale of barley yellow dwarf virus. phyoiathology 7g;g42- 96 946. of Cuthbertson" A. G. S., C. C. Fleming, and A. K' Murchie' 20fl3' Detection Rhopaiosiphuminstertum (apple-grass aphid) predation by the predatory mite Attystis' taiar"i v;mgmolecular gut analpis. Ag. ForestEntomol' 5:219'225' anq Gree,nstone,M. H. 1996. Ser;togicat-analysisof arthropodpredatiol: P$ fesent tutgre. pp. 265-300. fz W. O]C. Symondsonand J. E. Liddell [eds.] The Frology of Agricul-trlralPests - BiochemicalApproaches. chapman and Hall, Iondon. Greensltone,M. H., and A. K. Shufran. 2003. Spider predation: Species-specific ide,ntiication of gut contentsbypolymerase chain reaction. J. Arachnol. 3l: 13l-134. predatorgut Hagler,- J. R., and S. E. Naranjo. 1997. Measwing the sensitivity of an indirect cont€NrtELISA: detectability of prey remains in relation to pr€dator species, ternperature,time, and meal size. Biolog' Cont.: Theory and Appl' in Pest Manag' 9:ll2-119. Harwood, J. D., S. W. Phillips, K. D. Sundedand,and W. O' C' Symondson. 2001' Secondarypredation: quantification of food chain errors in an aphid-spider-carabid systemusing monoclonalantibodis. Mol. Ecol. 10:2049-2057' LaRock, D. R., and J. J. Ellington. 1996. An integrated pest manag€,nentapproach emphasizingbiologicat control for pecan4hids. southwest.Entomol. 2l: 153-166. Liao, H. T., M. K. Harris,F. E. Gilstap, andF. Mansour. 1985. Inpact of naturalenenries on the blackmargined pecan aphid Monellia caryella (Homoptera: Aphididae). Environ.Entomol. 14: 122-L26. Lim, U. T., and J. H. Lee. L999. EnzymeJinked immunosorbentassay used to anallze predation of Nilapamata /zgens (Homoptera: Delphacidae)by Pirata subpiratians (Araneae:Lycosidae).Environ. Entomol. 28:1L77-1182. Memil, C. R., D. Goldman and M. L. Van Kenreir. 1983. Simplified silver protein detectionsand image enhancementmethods in polyacrylamidegels. Electrophoresis3: 17-23. Platniclq N. 1974. The spider family Anyphaenidaein Arnerica north of Mqrico. Bull. Mus. Comp. 7nol. 146: 257-258. RichmarUD. 2003. Spiders(Araneae) of pecanorchards in the southwesteinUnited States and their role in pest suppression.Southwest. Entomol. Suppl. 27: ll5-123. Riechert, S. E., and T. Iockley. 1984. Spiders as biological control agents. Ann. Rev. Entomol.29:299-320. Sunderland,K. D. 1996. Progressin quantifring predationusing antibodytechniques. pp. 419-455. /z W. O. C. Slmondson and J. E. Liddell [eds.] The Ecology of Agriculturat Pests- BiochemicalApproaches. Chapmanand Hall, London. Sunderland,K. D., N, E. Crook, D. L. Stacey,and B. J. Fuller. 1987. A study of feedingby pohhagous predarorson cerealryhids using ELISA and gut dissection J. Appl. Ecol. 242907-933. Symondsorl W. O. C., M. L. Erickson, J. E. Liddell, and K. G. I. Jayawardena.1999. Amplified detectionusing a monoclonalantibody, of an aphid-specificepitope exposed during digestionin the gut of a predator. InsectBiochem. MoL Biol. 29:873-882. Tedders, W. L., and B. W. Wood. 1985. Estimate of the influence of feeding by 'Monelliopsis pecanis nd Monellia caryella (Homoptera: Aphididae) on the fruit, foliagg cartohydrate r€serv€sand tee productivity ofmature 'Stuart'pecans. J. Econ. Entomol.78:642-646. Wood B.W., W. L. Tedders,and J. D. Dutcher. 1987. Energy drain by threepecan aphid species (Ilomoptera: Aphididae) and their influence on in-shell pecan production. Environ.Entomol. 16: 1O45-1055. 97 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 occuRRENcE^N"ffiffi#B"r?:Nffi fJilit-ULLUSLANATUS) Mahmut Dogramaci,Wenhua. Lu, B" WarrenRoberts, Jim W. Shrefler,Mark Payton, Menitt J. Taylor,and J. V. Edelson Wes Watkins Agricultural Researchand Extension Center OklahomaState University, Lane, OK 74555 ABSTRACT Populationsof the squashbag, Anasa lrisfis @eGeer) (Hemiptera: Coreidae),and the cucumber beetle species complex, Acalymma vitatfiorr (Fab.) and Diabrotica undecirnpunctatahawardii Barber (Coleoptera:Chrysomelidae), were more abundantin time and space on watermelon, Citrullus lanaus (Ttrunberg) Matsumura and Nakai (Cucurbitaceae),than the melon aphid,Aphis gossypiiGlover (Hemiptera:Aphidae), or the spider mite speciescornplex, Tetrarryclwsspp. (Acariformes: Actinedida: Tetranychidae). Abundanceof the squashbug and the cucumberbeetles varied among geographicdishicts, among locations within a district, among peripheral and interior positions within a field, and among years. Squashbugs and cucumberbeetles occurred about a week earlier in the southeastand southwestdistricts than the northeastdistrict in the spring 1998. Populations of both insects were more frequent in time (sampling interval) or in space (number of fields) in the southeastdistrict thanthe northeastor southwestdishicts in 1998,1999, and 2001. Seasonalpattems of occurrenceof both insectsmay be related to planting date and initial abundanceearly in a growingseason. INTRODUCTION Watermelon, Citrullus lanatus (Thunberg)Matsumura and Nakai (Cucurbitaceae), is an important crop in the southernregions of North America. Approximately 16,000 hectares (40,000 acres) are grown in the south central states of Texas and Oklatroma (USDA 1999). The crop is valuedat approximately$1,000 per acrefor a total estimated annual value of g+g,g90,*O in the south central states,where similarities in production practices and pest problems have been documentedon an area-wide basis (Riley et al. 1998). Previous researchand grower surveys indicate that the major arthropod pests of watermelonare the squashbug, Anasa rnsrris(DeGeer) (Hemiptera: Coreidae);a complex of the striped and the spoftedcucumber beetles, Acalyrnma vitumrm (Fab.) and Diabrotica undecimpunctatahowardii Barber (Coleoptera:Chrysomelidae); the melon aphid, Aptrds gossypiiGlover (Homoptera:Aphidae); and the spider mite speciescomplex, Tetmychus spp. (Acariformes:Actinedida: TeEanychida€) (Quaintance 1899, Foster and Bnrst 1995, Pair 1997,Robinson and Decker-Walters1997, Riley et al. 1998,and Webb et al. 2001). Insect pests require control when pest density reachesan economic threshold. Pesticides r€present10-25% of the cost of watermelonproduction (Lu et al. 2003b). 99 Research on waterrrelon pest managementin oklahoma has been conducted primarily in laboratories,greenhouses, and experimentalplots in southeastemOktahoma (Bonjouret al. 1990;Edelson et aL.2N2,2003;Lu et al. 20o3a). of the pestsrecorded on watermelon,the squashbug andcucumber beetles are key speciesin the southcentral states (Riley et al. 1998;Foster and Brust 1995;Edelson et aI.2N2,2003). Thereis a lack of documentationin the scientific literature on occunenceof these pests on watermelonin commercialfields and their abundancein the southcentral states. Our specificinterests were in seasonaltrends in arthropodoccurrence in Oklahoma,and variation in abundance amonggeographic areas, fields within a geographicarea, or positionswithin a field. We report here results of surveys of these pest species and their temporal and spatial distributionson watermelonin Oklahomaover the periodsof 1998-1999and2}0l-20Q2. MATERIALS ANDMETHODS We conductedan extensivesurvey during a two-yearperiod of 1998and 1999that includeda total of 102 commercialfields (averagesize L2 ha) in 22 of the 77 counties, covering up to 3OVoof the watermelonproduction area in Oklahoma(Lu et al. 2003c). The evaluationof the arthropodpests was descriptiveand qualitative due to the extensivenature of the survey. An intensivesurvey during a 2-yr period of 2001 and 20[t2 wasconducted in both commercial fields and experimentalplots from different geogaphic areasor distant fields within the samearea but in a mannerto quantify abundanceand verify the statusof majorpests identified from the extensivesurvey. ExtensiveSumey. During 1998 and 1999, we surveyed 39 and 62 fields, respectively. The Oklahoma CooperativeExtension Service (2002) divides the state into four administrativeand geographicdistricts: northeast (NE), northwest(NW), southeast (SE), and southwest(SW). There was substantialwatermelon production in each district exceptNW. The 2-yr survey primarily coveredNE, SE, and SW following Lu et al, (2003c). We arbitrarily selectedthree sections(each l00m long and one row wide ranging from 0.9-7.3m)within eachfield. The sectionswere positioned such that therewere two near each periphery of a field and one in the interior. Within each section we used six transectlines, evenly spaced,with eachline startingat the baseof a plant in one row and extendingtowards the next row of plants, but ending midway betweenrows. The transect Iine was 3.7m long. We visually examinedplants along each of the six transectlines and rankedthe density of mixed adults and nymphsof squashbug, adults of the cucumber beetles,mixed adultsand nymphs of the melonaphid, and mixed adultsand nymphs of the spidermites as follows: 0 = no presencs,| = presentwith low densityof 1-9 individuals,2 = presentwith moderatedensity of 10-30individuals, I = presentwith high densityof >30. The densityranking was designedfor convenientfield assessmentand was not basedon economicthresholds for each of the arthropodgroups. This survey thereforedocumented temporaland spatial distributionsof thesearthropods in terms of relative abundance. Planting datesvaried statewideand planting occurredmostly in May in both years (Lu et al. 2003c). We begansampling as early as two weeksafter planting' Samplingin both yearswas initiated during the last two weeksof May and continueduntil the first week of August or the last harvest,whichever was earlier, at an approximateinterval of once a week. Samplingwas delayedin NE for oneweek, to lateMay, in 1998and three weeks to early June,in 1999 and was not as frequentin early growth stagesns in SE and SW because of late planting and cooler spring temperatures.There were thereforefewer fields sampled in the early and late intervals, In order to measurevariation in relative abundanceamong fields and within a field, we calculatedfrequency of arthropodoccurrence for eachsPecies over either total number of fields surveyedor total numberof transectsin which we noted occurrence of each arthropod species during the entire 12-wk sampling period' t00 Additionally we summarizedthe databy year and geographicdistrict. Due to the variation in planting date, *" measuredarthropod occurrence chronologically with samplinginterval' Uui aiO n-ot relate arthropod occurence to plant phenology among geographicdistricts' Therefore,therewere3districtsx3sectionpositionswithinafieldx6transectlinesxT-12 sampling- intervals in39-62 fieldseach year. intensive Sumey. We surveyedfewer fields in 2001 and 2002 but assayedmore intensivelywithin a field than in 1998and 1999. Fieldsof similar size (approximately0.4 ha) and cultural practices (such as fertilization and irrigation) were selectedeach year at differentlocations. In 2001,one field was locatedat Caneyand two fields at Lane,all in Atoka County in SE, and one field each was located at Fort Cobb in Caddo County and at El Reno in ianadian County,both in SW; watermelonwas direct-seededin the first and third weeksof May at Caneyand Lane, respectively, and the first andsecond weeks of June at Fort Cobb and El Reno, respectively. ln 20O2,one field each was located in Atoka County, Bryan County, and love County, all in SE; watermelon was transplantedin the first week of May. Growers involved agreed to apply no treatments for controlling arthropodpests each year. We divided eachfield into 16 samplingplots, each llx2Om in size. Plots were labeledby position within a field as comer (4), edge(8), and interior (4) to enableus to evaluate whether there were differences in occurrenceand abundanceof insects among these positions. Within each plot, three randomly selectedplants were visually examined for numbersof squashbug adults and nymphs and cucumberbeetle adults, approximately once a week from the beginning of the seedlingstage until the first week of August 2001 and at leastonce a week until the end of June2002. Our basicunit of samplingwas the areaof initial plant spacingof 0.9x3.7m(plant by row space).During early growthstages, we examinedthe wholeplant and the soil surfaceimmediately beneath it within the 3.33-m" area. In the later growth stagesafter the fifth sampling interval, when plant vines were intertwined witl one another,we included all foliage within the 3.33-m" areabeginning at the base of a plant and extending one half the distancetowards neighboring plants and rows. Therefore,there were 3 positionswithin a field x 3 plantsx 5-9 samplingintervals in three-four fields each year. We transformedthe data taking the squareroot of eachdatum plus 0.5 and comparedmean values for eachinsert at differenttimes of a growingseason on a per areabasis for effects of insect abundanceover time (sampling interval), between fields (geographiclocation), and amonglocations within a field (position). A linear model using PROC MDGD (SAS krstitute Inc. 1999) was tested for the three effects and their interactions(number of insect = samplinginterval I geographiclocation I position) at a significanto level of P = 0.0050,with a repeatedmeasurement procedure using sampling interval as the REPEATED factor and plant (position x geographic location) as the SUBJECTfactor. RESI'LTS Ertensive Surttey. Populationsof all four arthropodgtoups were found in fields in both 1998and 1999 (Frg. 1). Overall densityranking per transectper samplinginterval (meantSE) indicated that squash bugs (0.0334$.0021) and cucumber beetles (0.0172$.0015)were more than twice as abundantas melon aphids(0.0072t0.0010) and spider mites (0.0009$.0003) over the 2-yr period. Squashbugs and cucumberbeetles weremore abundant in 1999than 1998(Fig. I, legendbox). In contrast,melon aphids were more abundantin 1998and spidermites were generally low for both years(Fig, I, legend box). No populationsof any of the four arthropodgroups were detectedbefore the fourth week of May (Fig. 1). They becameabundant between late May and early July, and were 101 sotAsHBuG ltiE{ol2s BUG :l,g9 irrc+.ooeo 1998 lose+.orzo oSE{.035o 2 0.n 2 o.zz 0,18 = j o.re f; o.rs S o.ts i o.oe > 0.09 o.u 2 20.* o.oo B H 0.00 1 2 3 4 5 6 7 8 I 101112 123456789101112 !VEE( WEEK (' I o.ro z 0.08 f o.oe > 0.04 E o.oz fr ooo o 12545678910fi12 1 2 3 4 5 6 7 E 9 101112 WEEX wEl( Z. ^a^ = 0J4 j o.rr 2. o.rr # o.oe E o.oe > 0.06 > 0.06 A o.og 6 0.00 fr o.oo fr o.m 1 2 3 4 5 6 7 I I 101112 o 123456789101112 wE( litE( (, j o.ors = 0.012 f ooos I 0.006 6 0.003 fr o.ooo 23456789101112 o 123456789101112 WEK WEEK FIG. l. Relative abundance(mean density ranking per transect/ samplingintervd) of mixed adults and nymphs of the squashbug, adultsof the cucumberbeetle speciescomplex, mixed adults and nymphs of the melon aphid, and mixed adults and nymphs of the spider mite speciescomplex in watermelonfields from the third week of May (We.ek1) to the first week of August (Week 12) amongthe NE, SE, and SW districtsduring 1998 and 1999 when samplingbegan in the secondand fourth weekin NE, respectively.0 = no presence,I = l-9, 2 = L0-30, and 3 = >30 individuals. Overall meansin legend box rcpresent2,574 samples from 13.0fields per districtin 1998and 3,350 samples from 20.6fields per districtin 1999. 102 absent in the first week of August during both years. Regardlessof abundanceand geographiclocation, populations of all fourarthropod groups peake{ earlier in 1998 than iss6; .qu"stt uugs and-cucumber beetles occurred more frequently in time (sampling interval) than melon aphidsand spidermites in both years' The seasonalpattem of occlutence of the squashbug was similar to that of the cucumberbeetles that populationsof both insect groupsexhibited two peals in 1999,one in early and anotherin late-June,except that populationsof the cucumberbeetles in SE had only one peakin late June. Populationsofboth insectgroups in SE or SW peakeda week eariier than thosein NE in 19b8,even when plantswere available(Fig. l).The seasonal pattern of occurrenceof the melon aphid differed greatly from that of the squashbug and the cucumberbeetles (Fig. l). Populationsin SE peakedearly in the last week of May, but thosein SW peakedin the middle of July in 1998,about a 7-wk delay. Comparedwith those of the insects, populations of the spider mites were sporadic during most of the growing- seasonand absentfrom NB in both years(Ftg. 1). The numberof fields where we noled occurrenceof eacharthropod species differed among districts and this difference varied between years (fable 1). Squash bugs and cucumber beetles occurred in about 4OVoor more of the surveyed fields in both years. Populationsof the squashbug exhibited a similar spatial pattern as the cucumberbeetles each year: they occurredin more fields in SE and NE than SW in 1998,and more fields in SW than other districts in 1999. Melon aphidsoccurred in more than 4OVoof the fields in lgg8butfewerthanL0%inL999. Spidermitesoccurredinfewerthanl0%oof thefieldsin both years. TABLE l. Occurrenceof Four Arthropodsin WatermelonFields amongThree Geographic Dishicts of Oklahoma. ------r\iorra+ TotalNo. Tooffields@ fields surveyed Squashbug Cucumberbeetle Melon aphid Spidermite 1998 NE4 50 50 50 0 SE 20 45 )) 45 t0 sw 15 33 33 33 7 Statewide 39 4l 46 4l 8 1999 NE 14 43 36 7 0 sE 25 60 28 0 4 sw 23 65 56 t7 4 Statewide 62 58 40 8 3 The number of transectsin which we noted occurence of each arthropod species differed among positions within a field and this difference in within-field positions varied betweenyears (fable 2). Both years,populations of the cucumberbeetles and the spider mites were mor€ frequent at one edge or both edgesof a field than in the interior. Both years, this difference in within-field position was not as distinct for the squash bug. Populationsof the melon aphid were more frequent at both edgesthan in the interior in 1998but the spatialpattern was reversedin 1999when occunencewas low. 103 TABLE 2. Transectswith Occurrenceof Four Arthropods at Different Positionswithin a WaterrnelonField in Oklahoma. 7oof transects perposition Transect-position with arthropodoccurrence Squashbug Cucumberbeetle Melon aphid Spidermite 1998 Edge I 35 f) 42 t7 Interior 35 20 23 33 Ed;ge2 29 25 35 50 a Total No. transects 48 40 48 6 1999 Edge I 34 34 22 50 Interior 3l 25 M 0 Edge2 35 4L 33 50 Total No. transects" 219 r02 9 2 Total numberof transectswhere we found arthropodoccurrence, Intensive Survey. Over the 2-yr period of 2001 and 2OO2,both squashbug and cucumberbeetle populations were found in all fields exceptat Fort Cobbin SW; therefore, data from this location were eliminated from analyses. The overall number of insectsper areaper samplinginterval indicatedthat squashbug adults(0.6010.04 versus 0.30t0.03) andcucumber beetles (0.27t0.02 versus 0.0610.01) were more abundant in2Cf.2 than 2001, and that squashbugs were more abundantthan cucumberbeetles, with up to l1 adultsper arca. Nymphsof the squashbug (0.49t0.11versus 0,64t0.13) were similarbetween years. During 2001,sampling was started at differenttimes due to differentplanting dates, but at a growth stagewhen plants had three-eightleaves. All insect populationschanged in numbersignificantly over time and occurredmore frequently in time (samplinginterval) in SE than SW (Table3). However,no insectswere recorded in any fields for the first three samplingintervals except at Caneyin SE whereplanting was the eadiestamong all fields (Table 3). Adults of the squashbug appearedat Caneyearly in the first week of June, wherethe populationpeaked 2 weekslater anddeclined by the first week of July; nymphs of the squashbug were detectedapproximately 3 weeksafter detectionof adults at Caney where the populationpeaked late in the growing season(Table 3). Both stagesof the squashbug in other fields were noted later and reachedthe highestnumber at the end of the growing season(Table 3). Adults of the cucumberbeetles were not found at Caneywhere adultsof the squashbug occurredearly, but their populationswere noted and peakedin the first weekof July in fields at Lane andEl Reno,and reached another peak in the first week of Augustat Lane(Table 3). During 2002, sampling began when plants had two-four leaves. All insect populationsvaried in numbersignificantly over time (Table3). Adults of the squashbug and the cucumber beetles were detected as early as the first and second intervals, respectively. The former were found in all fields by no later than the third sampling interval(Table 3). Nymphsof the squashbug weredetected 4-5 weekslater andpeaked or leveled at the end of the growing season.Adult populationsof both insect groupsthat were recorded early in the first or second interval peakedin mid-May and again towards the middleof the 9-wk samplingperiod and declined at the last intervalin lateJune when fruits werernature (Table 3). Abundance of most insect groups varied significantly among fields in different locations,whether among geographic dishicts of SE and SW in 2001 or amongdifferent locations within SE in 2002 (Table 3). Adults and nymphs of the squashbug were more abundantin fields in SE thanin SW in 2001.and in morefields at Benninstonthan in other 104 o) EI c) nFFE:E:3 sEE TgrBf3!r o i?I x a e Els 6 EgEsE$Eg at) Hi.el g1F: rn E3Etl ()c) i*'$*i*i* i*'$ () qq00i F€ E€ (rt 3? r i ii $ r , ?? I I C) (f) $ rai \o o c.) ogq tto\ t: c) rO t-- \ci O O o rf-.) o RI I R8 -i ?E r+l i? '? ?TirE o r- o s€ I 8S So -.:j o -i U ;6 o c- G) 9888 F sE q ? iigAg EA?E ?EEIr o qgqs ooso q€- .;cjoo oo o E' € r85 9g (a g -i \o ??TiEEEEq.??E T t-- Gggd=dooo=Ri. & ooo s Eoo o a tr I 'e cn o 8BB I cl c oo @ 6l ???ETEEEEgEfiA E tt) b a 388o:oooo hoEo (.) 0) o odd E E v) g n9s 14 c{ 8888 8S?8oE?E U) q) oooo cjof o oSd +l \o oci N )< X o.l EE?I 555 oos ' 333' >\ I o z (\ s c € =)<)<, x)<)< EEY r YYY i Y!:- \o oos ct T (f) o Eq?r EEE,EEEr \o oog ooo o z c.i ql g c (n >.R 105 ll lxslFHTiBiFi5lj I-l3ri$jfif]iifl5 | ,-l ras ns8 ssRlF I lulsss s$s ssglE 'IISIHHHHFHiEgIF l*l$ae g$E$gslE € FII=I=g3gFEE3* EFEIF E Esl.l#Ei;Fff=lgs !E;.;;sggE;lF E g.l$lqgg 333"3331:"s ^^s 8 i l*lgE;EE3c==I€EE l;lEEs EEE e==lfr;E a) |-l=Eg EE= ;==l€*g F; | ;. tq rl r $l€gi s F EEI s,Es sEs s,EstrEF 106 SE fields in2.002. Adutts of the cucumberbeetles w€re more abundantin fields in SW than in SE in 2001, and in more fields at Lane than in other SE fields in2002. There were often (four out of the six analyses)significant interactions between the two main effects of samplinginterval and geographiclocation. Most insect gfoups were more abundantin peripheries(comer or edge) of a field than interiors in both yearsin most field locations,although this diffetence amongpositions within a field was only statistically significant for adults of the squashbug in 2001 (Iable 4). There were only three out of the 21 fields where interion had more insectsthan either TABLE 4. Number of Adults and Nymphs (mean*SE) of the SquashBug and Adults of the CucumberBeetle SpeciesComplex at Different Positionswithin a Field in Oklahomain ?-00Laad?.O02. Mean numberof insectsper area Fieldlocation Within-fieldposition Statistics Squashbug adult Lane A 0.93r0.19 0.61aO.08 0.33a{.08 '*Tf"jfliffi"' Lane B 0.23$.09 0.18rO.05 9:ii{:99 * -:' Caney 0.3010.11 0.21r0.w "0.2010.09 =offize El Reno 0.0310.03 0.03r0.02 ffi;- P Squashbug nymph Lane A 0.5610.34 1.18$.39 0.81lo.39 r wibir-fieldpocidm= v'+z L,aneB 0.99aO.51 l.ll$.57 0 5?aO.49 Caney o.73N.46 0.13l0.12 "0.r2j{.t2 ^ffi'- El Reno 0.00 0.00 P=0'6600 Cucumberbeetle adult Lane A 0.06aO.03 0.0210.01 0.07i0.04 F*irnio-nddp*u- =O'12 Lane B 0.06i0.03 0.M{.O2 0.03$.02 df =2,6?tt 0.00 0.00 0.00 Caney = 0.8864 El Reno 0.17$"06 0.18rO.05 0.1310.06 P 2W2b Squashbug adult Lane 0.7510.17 0.5310.09 0.56a{.14 F uorin-Reroposum = 0.91 Bennington 0.9510.16 0.8010.11 0.69{0.13 df =2,759 [.eon 0.1510.06 0.33$.07 0.73a{.16 P =0.4025 Squashbug nymph Lane 0.1510.10 [email protected] O.4l*0,37 Frioio-n npouio- = 0.27 Bennington 0.82rO.34 L.20t0.54 1.11aO.48 df = 2,907 Leon 0.00 0.1910.13 0.08$.05 P=0.7621 Cucumberbeetle adult Lane 0.4410.10 0.37rCI.06 0.39aO.09 F *;61a-6e6pp5166 = 0.37 Bennington O.25r0.Ul 0.1410.03 0.09t0.04 df =2,532 kon 0.19aO.06 0.26i0.05 0.31iO.08 P = 0.6886 " Watemplon was direot-seededin the first and third weeksof May at Caney and Lane in the southeastemdistrict, rcspectively, and in the secondweek of June at El Reno in the southwesterndistrict. b Watermelon was transplantedin the first we€k of May in all fields in the southeastem district. r07 comersor edgesover the 2-yr period. There were no interactionsof the two main effectsof samplinginterval and geographic location with the effectof within-fieldposition except for adultsof the squashbug in 2001 and2002when interactions between geographic location andwithin-field position were significant. DISCUSSION Our extensivesurvey was the first statewideinvestigation of major arthropodpests of watermelonin North America. The geographicareas, acreages, and fields this survey covered are collective representationsof watermelonproduction in the state in terms of climatic conditions,soil types,and managementpractices (Lu et al. 20Q3c).The intensive surveyprovided detailed data that werenot availablefrom the extensivesurvey. Thesetwo surveys complement each other and provide a better undentanding of occurrenceand abundanceof the four arthropodpest groups. Both surveysindicate that the squashbug andthe cucumberbeetle species complex occur throughout the major areas of commercial watermelon production in Oklahoma. Resultsof a 3-yr small-plotfield experimentin southeasternOklahoma (Lu et al.2N3a) agree with our findings that the squash bug and the cucumber beetles are the major arthropodpests of watermelon. Our resultsagree with studiesusing different cucurbit crops that the seasonaldevelopment of the squashbug largely varies amongdifferent geographic locations@onjour andFargo 1989). Both surveysindicate that populationsof the squash bug and the cucumberbeetles can occur more frequently in time (samplinginterval) in SE and SW than NE, or in SE than SW. Pestmanagement efforts shouldbe focusedon these insectsand geographic locations, Both surveysindicate that adults of the squashbug and the cucumberbeetles can occur in waterrnelonfields as early as one week after planting and the seasonalpattern of occwrence is bimodal wheneverthe insects are abundantand appearearly in a growing season;usually a low anda high peakoccur in earlyand late June. Whenpopulations in the spring were less abundant,we observeda unimodal peak in the seasonalpattern of either insect group, regardlessof geographic location. Lu et al. (2OO3a)consistently found a unimodal seasonalpattern that adults of the cucumber beetles peaked in midseason; populationsof mixed adultsand nymphsof the melonaphid peaked towards the endof the growing season,and populationsof mixed adultsand nymphs of the squashbug weremost abundantin the later halfofthe growingseason. Our resultstherefore agree with findings by Lu et al. (2003a) for the melon aphid, but do not completely concur with thein for the squashbug and the cucumberbeetles because of the bimodal peaks of abundancein early Juneand the endofJune in someyears. Both surveysalso agree with prior findingsthat adultsof the squashbug on squash plants generallyreach greatestabundance at flowering and fruit setting stagesregardless of plantingdates (Fargo et al. 1988,Palumbo et al. 1991). Abundantinsect populations at the end of June and beginningof July ,ue not expectedto causesignificant reductionsin watermelon yield since fruits reach maturity and can be harvestedin one or two weeks @delsonet al. 2003, Lu et al. 2ffi3a). However,high densitiesof the squashbug can cause significant mortality of watermelonseedlings at the vining or flowering stagesand therefore reducefruit yield @delsonet ^1.2W2,2003). The cucumberbeetle species complex can causesignificant damage to watermelonseedlings in the eady season(Quaintance 1899, Fosterand Brust 1995). Reducinginsect pest populations in late crop seasonmay serveto derreaseearly seasondamage to watefinelonin the next year. The intensive survey indicates that planting date and planting method may have significant effects on abundanceof both the squash bug and cucumber beetles on watermelon. A greater abundance of both insects occurred in watermelon when. 108 transplantingoccurred early in 2Cfl2as comparedto the later planting dates in 2001; squash bugs-appearedapproximately four weeksearlier in the eady-plantedfields than in the late- pta:ntedhetdsin Z-OO1. This suggeststhat damagefrom overwintering squashbugs could be avoidedby delayingplanting dates. notn surveysindicati that squashbugs and cucumberbeetles occur more frequently in field peripheries(corner or edge) than in interiors. This does not necessarilycorrelate with arthropbdabundance, suggesting that field orientationwith respectto prevailing winds or sunlightmay be importantfor insectdispersal. Our findingsagree with Morishita(1992) thatthere are higher densities of spidermites at field edgesthan in interiors' We conclude that results from the extensiveand intensive surveysindicate l) the more abundantsquash bugs and cucumberbeetles are in time, the more widely they occur in geographic areai2) both insects occur most frequently in SE and more frequently on peripheriesof a field than in interiors; 3) both insects can occur as early as a week after planting and reach their highest densitiesin the later half of a growing season;and 4) the seasonalpattern of occrlTence varies relative to arthropod species, planting date, or abundanceearly in a growing season. ACKNOWLEDGEMENT We thank growe$ for participating in the surveys and collaborating with data collection, and are grateful to the Oklahoma Cooperative Extension Service for identification of growers. This research was funded in part through the Oklahoma Agricultural Experiment Station, the PestManagement Alternatives Prograrnof the USDA CooperativeState Researchand Education Service (CSREES),the OklahomaCooperative ExtensionService, and the USDA/CSREESSpecial Grants Program. LITERATIJRE CITED Bonjour,E. L., and W. S. Fargo. 1989. Host effectson the survival and developmentof Anasatristis (Heteroptera: Coreidae). Environ. Entomol. 18: 1083-1085. Bonjour, E. L., W. S. Fargo, and P. E. Rensner. 1990. Ovipositional preferenceof squash bugs (Heteroptera:Coreidae) among cucurbits in Oklahoma. J. Econ. Entomol. 83: 943-947. Fdelson,J. V., J. A. Duthie, and B. W. Roberts. 2002. Watermelonseedling growth and mortality as affected by Anasa rn'sfr's(Heteroptera: Coreidae). J. Econ. Entomol. 95: 595-597. Edelson,J. V,, J. A. Duthie,and B. W. Roberts,2003. Watermelongrowth, fruit yield and plant survival as affected by squashbug (Heteroptera:Coreidae) feeding. J. Econ. Entomol.96:.64-7O. Fargo,W. S., P. E. Rensner,E. L. Bonjour,and T. L. Wagner. 1988. Populationdynamics in the squash bug (Heteroptera: Coreidae) - squash plant (Cucurbitales: Cucurbitaceae)system in Oklahoma.J. Econ.Entomol. 81: 1073-1079. Foster, R. E., and G. E. Brust. 1995. Effects of insecticidesapplied to control cucumber beetles(Coleoptera: Chrysomelidae) on watermelonyields. Crop Prot. 14: 619-624. Lu, W., J. V. Edelson,J. A. Duthie, and B. W. Roberts. 2003a.A comparisonof yield betweenhigh and low intensity of crop managementfor three watermeloncultivars. HortScience38: 351-356. Lu, W., J. A. Duthie, B. W. Roberts,M. J. Taylor, and J. V. Edelson. 2003b. Partial budget analysis of effects of crop managementintensity on profitability of three watermeloncultivars. J. Veg. Crophod. 9: 49-71. 109 Lu, W., B. W. Roberts,J. A" Duthie,J. W. Shrefler,M. J. Tayloa andJ. V. Edelson.2003c. characteristics and geographic variation of watermelon (citrullus laruns) productionin Oklahomafrom a statewidesurvey. J. Veg. Crop prod. 9: 334g. Morishita, M. 1992. Movement of 2 speciesof tetranychidmites (Acarina, Tetranychidae) from bordervegetation to watermelonfields. Jap.J. Appl. Entomol. zaol.36: 25- 30. Oklahoma Cooperative Extension Service. 2W2. Administrative disticts and area specialists,pp. vl-25. In 20a2 PersonnelDirectory. oklahoma state university, OklahomaCity. Pair, S. D. 1997, Evaluationof systemicallytreated squash trap plant and attracticidalbaits for early-seasoncontrol of shiped and spotted cucumber beetles (Coleoptera: Chrysomelidae)and squashbug (Hemiptera:Coreidae) in cucurbit crops. J. Econ. Entomol.X): 1307-1314. Palumbo,J. C., W. S. Fargo, and E. L. Bonjour, 1991. Colonizationand seasonal abundanceof squashbug (Hemiptera: Coreidae) on summer squash with varied plantingdates in Oklahoma.J. Econ.Entomol. 84l.2?tl.-229. Quaintance,A. L. 1899. Someimportant insect enemies of cucurbits. Geor.Exp. Stn. Bull.45: 25-50. Riley, D. G., J. V" Edelson,R. E. Roberts,N. Roe,M. E. Miller, G. Cuperus,and J. Anciso. 1998. Integratedpest managementfor cucurbit crops in the south-centralUSA: pest status, attitudes toward IPM and a plan for implementation. J. Extension 36: Featurearticle 3 (http://wwwjoe.orflodl998august/E.html). Robinson,R. W., andD. S. Decker-Walten. 1997. Cucurbits.Crop ProductionScience in HorticultureSeries 6. CAB Intemational,New York. SAS InstituteInc. 1999. SAS/STATUser"s Guide, Version 8. SAS InstituteInc., Cary, NC. USDA. 1999. AgnculturalStatistics 1999. GovernmentPrinting Office, Washington,DC, Webb,S.8E., D. G. Riley, andG. E. Brust. 2001. Insectand mite pests,pp. l3L-149. InD. N. Maynard[ed.], Watermelons: Characteristics, Production, and Marketing. ASHS Press,Alexandria, VA. ll0 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 ADT LT WHITEFLIES (I{OMOPTERA: ALEYRODIDAE), Al.lD WHITEFLY PARASITOIDS (I{YMENOPTERA: APIIELINIDAB) RESPONSETO COOL WHITE FLUORESCENT LIGIIT POWEREDBY ALTERNATING OR DIRECT CTJRRENT C. C. Chut, T.-Y. Cheq and T. J. Henneberry USDA ARS, PWA WestemCotton ResearchLaboratory 4135E. BroadwayRoad Phoenia AZ 8504G.8803 ABSTRACT Attraction of Bemis nbaci biotype B (Gennadius), Trialeurdes vqorariorum (Westwood), and Trtaleurdes abutilonea (Ilaldeman) whiteflies and Eretmocerus eremictrs@ose and Zolnerowich) and Encarsiafomosa (Gahan) whitefly parasitoidsto alternating or direct electrical current powered light sourceswas studied in a dark room. Fewer adults of all whitefly specieswere caughton Tanglefoot@coated transparent plasic cards placed between a whitefly releasechamber and the direct current powered white fluorescent lights compared with alternating curent powered fluorescent lights. Differences in catchesof whitefly parasitoidsin responseto lights poweredby alternating or direct current were not significantly different. INTRODUCTION Several whitefly species are economic pests of field and greenhousecrops worldwide(Mound andHalsey 1978, Gerling and Kravchenko 1996). Ofthe Aleyrodidae, Bemisia abaci (Gennadius) biotype B, Trialerrodes vqorariorum (Wesrwood), and Trialeurodes abutilonea (Ilaldeman) are the most common economic greenhousecrop pests(Liu and Oetting 1993). Under greenhouseconditions B. abaci hasbeen partiarlarly damaging in recent years. Integrated pest manaSement@M) systemswith focus on biological control have beenof particularinterest (lloelmer and Kirk 1999). Most IPM systems in greenhousesrely on yellow sticky card or other traps to monitor whitefly populations. When light flicker 1= ripple) of alternatingcunent (AC) poweredlight, a physical phenomenon,is modulated,the visual flicker effect changessimultaneously. Flicker fusion frequency(FFF) is the ftequencyabove which the eye cannot detectthe flicker changebecause it exceedsthe rate at which th€ eye processespictures. It is known that insectsand human eyes differ significantlyin spectralsensitivities. Humans have a FFF on the orderof 50 hertz(tlz), while someinsects appear to respondto frequenciesupwards of 300 Hz (Shields1989). Shields(1980) reported that minutepirate bugs, Orius tristlcolor (Whhe), travel distancedecreased and turning ratio increasedas the flickers in the AC light was reduced. Syms and Goodman(1987) reportedthat an insect trap with a flickering UV electrocutorlamp caughtthree times as manyreleased houseflies, Musca domestlcaL., as trap poweredwith non-flickerUV lamp. Miall (1978) mentionedthat l-acusta mlgrotoria (R andF), andfive otherinsect species oould resolve 6o/o 100 IIz flickers. '[email protected] 111 - Our objective in the current work was to detenninethe attractive responseof 8. tabaci, T, vqnrariorum, and r, abutilorca and their parasitoidsEretmocerzs eremians @oseand-Zolne-rowich) nd Encwsiafumov (ciahan)to white fluorescentlight powered !t lc electricity compared with direct current @c) electricity light sourcei in the laboratory. The purposeof the study was to developan insect trap for use in greenhouse farming as a survey and monitoring tool. MATERIALS AI{D METIIODS The light bioassayapparatus (Fig. l) consistsoffourlateral branches(A), attached at right angles to each side of a square,centrally located insect-releasechamber @), Lateral branchesand the releasechamber are constructedof 2-mm thick clear Plexiglasil. Lateralbranch dimensions are 30+m long x l2-cm wide x l2-cm high-opposite rezulting in 144- cm2 1C; openings on each end. At the ends of each lateral branch, the insect- releasechamber, connectors made of black Plexiglas @) are attachedto the distal endsof the lateral branch and light source compartments@). The conn@torshave overlapping Plexiglas pieces (F) that extend beyond the connector ends to accommodatethe eroct outside dimensionsofthe lateral branch on one end and the light sourcecompartment on the other end. FIG. l. Light bioassaysystem for studying whitefly and parasitoidresponse to alternating and direct electrical current poweredcool white fluorescentlight. The insect-releasechamber (Fig. 2) hasfi29-cnf (27 x27-cmoutside dimensions) clearPlexiglas cover plate (Bl) with a centrallylocated knob @2). The body sides@3) of the compartmentare l2-cm wide x 12.4-cmlong with 6.8-cm wide and l2-cm long openingsinto the lateralbranches. The @mersof the body sidesare double layeredto form recesses@4) accommodating9.5-cm wide x ll.S-cm long x 2-mm thick black plastic panelsto closeoffthe lateral branchesfrom the insect-releasecompartment prior to insect release. Number 103 Tough Frost light difhsion filters @oscolux, Rosco, Stamford,CT) are locatedin the connectorsat the junctions ofthe connectorsand light source to distribute the light evenly into the lateral branches and insect-release compartment(Fig. 3, Dl). Removableblack plasticfilm holders(13-m long l2-cm wide) n2 FIG. 2. Detailsof the insectflight releasechamber component of the light bioassaysystem for studyinginsect light responses. c3{ FIG. 3. Detailed drawing of lateral branch and light source connectorFig. lD and film holderFig. lG. ll3 (Fig. 3, Gl) are insertedin the centersof the connecton (G3). The film holders have 9.5- cm x 9.5-cm rectangularreoesses, that accommodate0,5-cm thick black plexiglas ftames (1011-91x 10.7-cm),which hold 9.5-cm x 9.5-cm clear plastic-backedtheatrical clear ngel"-liglrt_filtef "glr (Fig. 3, Gz). The Theatrical clear fifters @oscolu:r, Rosco, Stamford, CT) face the insect-releasechamber. The filters are coated with Tanglefooi (Tanglefoot@Co., GrandRapids, MI) to catch insec{sattracted to the light sources. Light intensities are measuredwith a pyranometerconnected to a light meer @icor 100, Lic-or, Inc.,Lincoln, NB). The aluminum light box oomponents(E) weip purchasedaom epw Enclosures(Salt Lake city, LIT). They are 14.5-cmlong with 12.5-cmx 12.5-cminside diameter oPenilg (156-cm2 areas). Interiors are paintedwhite and exteriors black. Ligtrt sourcesare 13 w, 2'l0o\" 900 lumen compact fluorescentlamps (cFL) (Sylvania Dulux cFl3DD/E/827, osram sylvania ProductgInc., Maybroob ND, poweredwith solid state balldsts. For AC operatio4the solid stateballast (Fig. lll FulhamHigh power, class p, PEPIOO,120V,.60 AC input, 0.15 ampq Fulham,Inc., Ilawrhorng CA) wereused. These ballastswere found to be insufficiently filtered, allowing some 120 rlz ripple to appearin the lamp output. A DC-operatedsolid stateballast (Model # SHl2-13, l2V DC input, 1.2 amps,Light-it Technologies,Dilloq MT) designedfor recreationvehicles and boating applicationsprovided ripple'free light. They are poweredby small regulatedDC power supplies(Lambda Model LLJD 15-33,output 12 V, PartsExpresg Springboro,OtI). A simple l2-bit parallel-portdata acquisitionsystem @r. chris Braun,colorado Schoolof Mines, Golden,CO) was usedto gatherdata of the light output of lampsoperated with both ballasts.Waveforms of CFL outputsfor the AC andDC ballastsare shownin Fig. 4. The rippfe(ratio of true root-mean-squarevoltages to the DC vohage)werc29/o and0.T/o for the AC andDC solid stateballasts powered CFL sources,respectively. Lateral branch extensions(Fig. lD with 2.7-cm x 2.7-cmscreen covered openings for ventilationwere optional additionsfor insectlight studiesover longer distances. 3. I o nME(mnrseo) FIG. 4. Waveforms of alternatingcurrent solid state(waved) and direct current solid state (straight) vottages. B. labaci were obtained from cotton fields and reared in an insectary that had a mi*ture of cottoq Gossyipium hirstum L., and several vegetabte crop species. T, vaporariorum were obtainedfrom a commercialstrauberry greenhouseand provided by J. L. Bi of University of Californiq Riversidg CA. They were rearedin a temperature controlled growth chamber(12 h dayll2 h night; 25"120oC). T. abutilonea was obtained from an alfalfa field in Calipatria"CA and rearedon cottonin a greenhousc.E. eremlqts andEn. lormov were obtained.ftom SyngentaBiolines (formerly Norvatis BCM North Americq Oxnard,CA), tl4 The experimentaldesign was a randomizedblock with trvo treatmentsand ten replicates. The treatmentswere AC and DC electricity powered cool white fluorescent tights. For eachinsect species described, one-hundred adults were placed in a cappedglass vial for eachreplicate trial. The vial with insectswas placedin the releasecompartment under dark conditions. The vial was uncappedto releasethe insectsand the cover plate set in place. The black plastic panelsclosed off the lateral branch entrancesto the light sources. A white fluorescentlight was placedon the top of the insect-releas€compartment to stimulatethe movementof adultsout of the vial (Chuet al. 1998). In operatiorqlights at the end ofthe lateralbranch compartments were activatedand the black plasticpanels at the lateral branch entrancesof the insect release compartment were removed, which allowed the adults in the releasingchamber to dispersein responseto light sources. The Tanglefoot-coatedtransparent films were replaced24 h following releasesand the numbers of insectswere counted. Ambient temp€raturesduring the studieswere 26.1fr.4 to "C 30.11{.2,32.4fl.O,30.710.1, 28.0fr.2, and26.5f0.8 for B. tabaci,T. ryortiorum, T. abutilonea,E. eremicus,and En. formosa, respectively. Numbers of insectsfapped for AD and DC powered light treatmentsin each replicatefor each.insect specieswere averagedfor eachtreafinent and meansseparated using t-tests. RESLJLTSAI.ID DISCUSSION Mean numbers of adult B. labaci biotype B trapped on sticky cards were significantly greaterwhen the white fluorescentlight was poweredby AC than by DC when distances from the insect-releasepoint were 3q 60, or 9Gcm (Table l). Mean numbers of adult T. ryorariorum and T abutilonea trapped on sticky cards were also greater when the white fluorescentlight was AC comparedwith DC power. Mean numbersof adult E eremic'usand h. Formos trapped on transparentsicky cards were not influenced by AC or DC power sources. It apperrsthat tne small (less than lYr) 2Ok tlz frequencyripple associatedwith the DC poweredCFL was far too high to elicit vision responlreof whiteflies but not whitefly parasitoids. It renrainsunerglained why whitefly parasitoidscould respondto suclt high frequency ripple. The biological and behavioral implications ofthe different responsesof speciesare also unknown. TABLE l. Mean t SEM Numbers of Mult Bemisia labaci Biotype B, Trialeurodes vaporarioram, Trialeardes abutilorca, Eretmeerus eremictts and fuarsia formov Respondingto Alternating(AC) andDirect Current@C) PoweredCool White Fluorescent Lights. Distance ftom light Light intensity Meanno. adultVtrap/24h' Species source.cm Wm2 ffi B. labaci 90 0.831 33.3t1.6a' l0.3tl.4b 67.4 60 1.419 26.8t3.1a 18.9t2.9b 4.4 30 1.770 31.5x2.6a l3.7tl.4b 56.5 T vaporariorum 60 1.224 25.8t2.5a 13.8+1.5b 50.2 T. abutilonea 60 1.395 28.9t1.0a 9.5+1.0b 142.4 E. eremicas 60 1.434 l7.2tl.4a 2l.4t2.la 2.6 En.formos 60 1.392 9.9l'1.5t lO.4x2.4a 1.7 o Meansof a pair in a row not followedby the sameletters are significantlydifferent (t- test,P = 0.05,df = l, 9). Light intensitywas measuredby Watts persquare meter (W\m2). 115 The results do show, howwer, that more B. tobaci, T vqorotorum and r. abuilonea yg9?ught in gr-eenhousan lgspondingto AC poweredwhiite fluorescentright comparedwith DC poweredfluoresc€nt fight sources. ACKNOWLEDGMENT authorsire parmer, sr-atefurto J. M. oprical Sciencescenter, University of ^-_^_]1" Tucsoq tt"ong AZ, for designof the modulatedlights and editing of the ealy versionof the manuscri{t. They also thank D. Hawker, weslern Area powir Administration, U.S. Departmentof Energy, Phoenix, Az andc. G. Jacksonfor their rwiew of the manuscript. Technical assistanceswere provided by Scott Davis, Tawnee wilbur, e-y oppo, a'na Otto Isensee.Figures were created by Lynn Forlow Jech. LITERATURECITED Chu, C. C., T. J. Henneberry,-an{ M. A. Boykin. 199g. Response of Bemisiaargentifotii (Homoptera: Aleyrodidae) adults to white fluorescent and incandescenitight in laboratorystudies. Southwest. Entomol. 23: 169-lg l . Gerling,D., and v. Kravchenko. 1996. pest managemertof Bemisiaout of doorq p. 667- !ll. In D. Gerling led.f Bemisia: 1995Taxonomy, Biology, Damage,Control and Management. Intercept,Andover. Hoelmer,K. A., and Kirk, A. A. 1999. An overviewof naturalenemy explorations and evaluations for Bemisia in the u. S. Bull. oILB/sRop (IoBcAil/pRs). 22: lo9- tt2. Liu, T.-X., and R. D. oetting. 1993. Morphologicaland developmentalcomparison of three whitefly speoies (rlomoptera: Aleyrodidae) found on ornamental plants. university of Georgia college of Agriarlnrral and Environmental Sciences, GeorgiaAgric. Exp, Stn. ResearchBull. No. 412,ll pp. Miall, R. c. 1978. The flicker frequenciesof six laboratoryinsects, and the responseof 'ripple'. the compoundeye to mainfluorescent Physiol.Entomol. 3: 99-106. Mound, L. A., and s. H, Halsey. 1978. whitefly of the world. A systemiccatalogue of the Aleyrodidae (Homoptera) with host plant and natural enemy data. sritistl Museum (Natural History), 340 pp. Shields,E. J. 1980. Locomotiveactivity of Orius tristicolor undervarious intensities of flickering and non-flickerlight. Ann. Entomol.Soc. Amer. 73:74-77. shields,E. J. 1989. Artificial light: Experimentalproblems with insects. Bull. No. 35, Entomol.Soc. Amer. Summer:4043. SymsP. R., and L. J. Goodman. 1987. The effeotof flickering u-V light outputon the attractivenessof an insect electrocutor trap to the housefly, Musca domestica. Entomol.Exp. Appl. 43; 81-85. hltdred bythe U,S.Deparlment of Agrlodture FOROFFICIAL USE ONLY ll6 vol,.29 NO.2 SOLNIWESTERN ENTOMOLOGIST ruN.2004 HYDROCARBONS OF GREEN A}ID YELLOW COLOR MORPHS OF COTTON APHIDS WITHIN AND ACROSSPOPULATIONS LeahannM. Borttr and Richard J. Deslippe De,partnentof Biological Sciences,Texas Tech University Lubbock.TX79409-3131 ABSTRACT We identified both @)-pfamesene and a total of 13 z-alkane hydrocarbonsfrom hexaneextracts of cotton aphids,Aphis gossypii (Glover), collected from Lubbock and Brazos Counties, Texas, and Kem County, California. Discriminant function analysis was used to differentiate the hydrocarbon profiles of green and yellow morphs of apterousfemales across geographically separated populations. The alkaneswere conrmon to both color morphs,but the relative proportionsof the compoundsdiffered significantly within each populafion, an unexpectedresult since the chemicals contibuting to the pigmentationof aphidsare independentof hydrocartons.Compared to the yellow morph, the gree,nmorph had reduced abundancesof almost every detected hydrocarbon for Lubbock and Brazos Counties, but had greater abundancesof almost every detected hydrocarbonfor Kem County. Among populations,hydrocarbons differed significantly not only in relative conce,ntations,but also in their t1pes,a result consistentwith studies on other groupsofinsects. INTRODUCTION Besidesacting as chemical barriersto the entry of pathogensand retardingwater loss (tockey 1988), lipids on the exoskeletonof insects are commonly involved in conspecificand heterospecifiqcommunication (Howard 1993,Singer 1998). Various tt- alkanes, alkenes, and methyl-branched components are typical components of the epicuticle, and they collectively provide a hydrocarbon(HC) profile. Becauseinsects synthesizecomplex profiles with many qualitativeand quantitativecharacteristics, the profiles reflect their genotlpes and are sometimesused in taxonomy (tockey 1988, l99l). Cuticular lipids of most aphids include normal and branched alkanes.Of the branched alkanes, methyl-branchedHCs are often the major component of cuticular lipids. However, the z-alkanes in some aphid speciesare consistentlythe predominant componeNrtof the cuticular HCs (Lockey 1988). For example, the dominant cuticular hydrocarbonsin the cereal ryhrd, Sitobion avenae(Fabricius), are ,-p€ntacosaneand t- heptacosane(Hebanowska et al. 1989), while n-pentacosane,z-heptacosane, n- nonacosan€and n-he'ntriacontaneare the main componentsfor the Russianwheat aphid, Diuraphis noxia (Mordilko) (Bergmanet al. 1990).As a result of the unique n-alkane profiles of aphid species,the use of theseprofiles can be used for aphid identification (Dillwittr et al. 1993). t_It7 cuticular HCs have proven especiallyuseful in researchin chemicalecology and systematicsofinsects includingdipterans (Bartelt et al. 19g6),orthopterans (Lockiy and oraha 1990), isopterans(Haverty et al. 1999),hymenopterans (Howard et al. 2001) and coleopterans(Lockey and Metcalfe 1988). Here we characteriie the n-alkane cuticular Hls of green and yellow morphs of apterous female cotton aphids, Aphis gossypii (Glover). Becaused ifferent p opulationso f t he s amei nsects peciis - "y h"o" d ifferent HC profiles (Howard 1993), we also compared HC profiier o.ort geographically separatedpopulations. The cotton aphid is an important crop pest distributed globally in tropical, subtropicaland warm temperateregions. This aphid is exhemely variuuti in color and size, and has been described under more than 40 synonyms all over the world. This morphological variability has caused many difficulties for taxonomists (kclant and Deguine 1994), and thus the characterization and use of their HCs to facilitate identificationcould complement standard taxonomic methodology. MATERTALS AND METHODS Apterousfemale, g reen a nd yellow I . g ossypii morphs w ere collectedi n I 998 from cotton fields throughout Lubbock and Brazos Counties,Texas, and Kem Countn Califomia. The two counties in Texas were separatedby about 833 hn. Aphid nymphs are smallerthan adults, and every effort was madeto collect large adult aphidsof similar sizes.For insectsthat have multiple generationsper year, seasonalinfluences on HC profiles can be important, so we collectedall of our samplesin Septernber.Because most aphids remain on one leaf for their entire life span (Slosser et al. 1992), ten aphids (representingone replicate)were collectedfrom one cotton leaf at eachcollection site. All te'naphids were placed in individual glassvials, rinsed in 70 pl of hexane,and then removedfrom the vials leaving only the aphid extract. Within 48 hr from collection, the aphid extractswere analyzedusing a GCA{S system(Hewlett Packard[HP] 6890 Series II Plus gas chromatographcoupled to an HP 5973 Seriesmass selective detector). After severaldozen trials on the GC/14Sto find the appropriatetemperatures and time method for our aphid extract samples,we settled on the following parameters,The oven was programmedfrom 100to 280"C at lO"C permin for 10 min, andthen 25oC per min for 3 min with a2-min pre-runhold and a 7-min post-runhold. The injection and detection temperatureswere 250oC and 230oC,respectively. Splitless injection was used, and sampleswere carried through a 5%-diphenyl-95%-dimethylsiloxanecopolymer capillary column(HP-5MS, 30 m X 0.25 mm ID, 0.25 pm film thickness)with a constanthelium flow at 1.2 ml per min. We acquiredmass specFa from 50 to 550 amu (2.94 scans/s),and used ChemStationsoftware (HP GI701AA) to integrate the area of chromatographic peaks. Analysesof aphid extractsby GC/IVISrevealed the presenceof many compounds. Retentiontime of each peak was determinedby comparisonwith known intemal z- alkane standards(Bedoukian Research,Inc.), and compoundscorresponding with the peaks were identified by comparison of molecular ion (M+) of known z-alkane standards.Chemical profiles were analyzedby taking the samepeak areameasurernents for each individual sample, giving rise to a series of chromatograrnsthat differed by relative abundanceand/or type of compound.Relative abundanceof eachcompound was calculatedas a percentageofthe total peakarea across samples. We used discriminant function analysis (DFA) to differentiate chromatogram profiles of aphidswithin and acrosspopulations. All DFAs were conductedwith cross- validation, a techniqueused to compensatefor an optimistic apparenterror rate (percent ll8 between of misclassified observations). comparisons of peak areas were made equalized iyo.o..ruon profiles by meansof z-scorefansformaiions. This transformation variability ttre effect of each variable by conecting for large differences in scale and h ydrocarbon amongt he p eak areas.A M ANOVA was p erformedo n t he q uantities.of packagesfor oeak areasafter the z-score transformationt.ff4i"ituf and Matlab statistics Wiodow.* were used. RESULTS from we detectedboth (E)-pfamesene(EBF) and 13, 12 and l0 n-alkaneHCs in Texas the hexaneextracts of A.-goisypit collectedin Lubbockand Brazoscounties andC29 ft 11glnCounty in califolrnia,iespectively(Fie. 1).Eachalkane betw-een Cl5 Cl9' was detectedin every population,with the ixceptiont of C23 and C24 for Lubbock' The longer chain C23 and C24 fot Brazos, and Ci9, C2l, C22, C23 and C24 for Kem' population(Fig' ."ipo*At betweenC2i ndC29 were the most abundantHCs in each 2). LS. r2 rf 11) e 2 3 4 sq zq t,.ltPtl 6 tt E ! t.4e147 \zct{il7 te+{)7 6c+{l6 &6 2*06 s6 .7 8J o t2r3l RetentionTime (min) peaks FIG. l. Typical chromatogramof whole body exfiacts of A. gossypii.Numbered roi"tponiito ttre followilg compounds:(l) (E)-B-farnesene[EBF], (2) Pentadecane (5) Octadecane (6) tClsl, (3) Hexadecane[C-16], (4) Heptadecane[Cl7], -[Cl8]' (?)-D-glosane{C221'(10) il;"fi;.;" [c19], (7) Eicosanetciol, (a) HeneicosanetC2U' ( 13)Octacosane i*toot*t iczsJ, i it; tt"*ooi*elC26l, ( 12)HeptacosmelC2T\, our intemal standard(I.S.) was [c2g], (14) i_rreiacosanat,(15) Nonacosane[c29]. tetRpo$me. ll9 0.80 0.70 I G.pen Morph (n = 171 0.60 ! YehwMorph(n=29) 0.50 0.40 0.30 0.20 0.10 0.00 0.80 g 0.70 I Greenwtorph (n = 10) F o.do Yettowltorph(n = € fl 10) = 0.s0 t o.4o 0.30 .g f 0.20 & o.ro 0.00 0.80 0.70 I GrcenMorph(n=5) 0.60 E YeUowltorph(n=8) 0.50 0.40 0.30 0.20 0.10 0.00 E io \o F € o\ c rr cr |o \c F € s\ EU66UUUUSUOU$S Hydrocarbon FIG' 2. Relativeabundance ofsaturated hydrocarbons ofgreen and yellow morphsofl. gossypii populations collected throughout Lubbock and Brazos counties, Texas, and Kem County, California. 120 The alkaneswere collxron to both the green and yellow color morphs, but the the compoundsdiffered significantly,withirreach population relative proportionsof = l" = 0.130;f = A.Sqg; df = 14, 3l; P < 0'001,Brazos: Wilks'tr' (Lubbock:Wittt' = O.Ztg,f = 13.468;df = 4, lj; P< 0.001,and Kem:Wilks'l'= 0'003;F:58'604; df |0,2;P=0.02)(Fig.3).FortheLubbockpopulation,DFAhadl0o%.conect I Lubbock a I a a a a 5 vr4 Brazos E: ! AL El t .Eo a .E -1 a ba I .A E-3 a 25 20 Kenr 15 10 5 0 -5 -10 -15 -20 Green Yellow Color Morph FIG. 3. Discriminant function analysis of green and yellow morphs of A. gossypii collectedthroughout Lubbock and BrazosCounties, Texas, and Kem County, Califomia' There were significant differences between the color morphs within each population: Lubbock(P < 0.001),Brazos (P < 0'001)and Kem (P < 0.02). t2l classificationof the samplesinto their respectivemorphs, and cross-validationresulted in an error of 6%. For the Braeos and Kem populations,DFA had 95%oand l00o/ocorrect classification,respectively, of the samplesinto their respectivemorphs. cross-validation did not alter the percentages.eompared to the yellow morph, ttt" gr"* morph-had reducedabundances ofeach ofthe detectedHCs eicept c29 for LubbJckandci5,c26 and c29 for Brazos. In contrast to the yellow morph, the gree,nmorph had greater abundancesof eachof the detectedHCs exceptcl5, cl6, c[a, cza ^i czs for Kern (Fie.2). A pooled DFA on the samplesfrom all three populations revealed significant = llfferencesacross populations (Wilks, l. 0.002;f = {Z.OSI;df : 70,289; p < O.OOI) (Fig' a)' Correctclassification ofthe samplesinto their respectivegroups was observed n 94% of cases.Cross-validation resulted in an error oi tSyo. tt" n"ti.lt"t orith " correlation of 0.7 or above for this DFA were consideredto contribute the most to the discrimination across populations and between color morphs. Those variables in descendingorder were Cl8, C25,EBF, andCl7. o Lubbock Grcen r Lubbock Yelbw o Brans Gr€en -Braas Yellow o KernGreen il2 t Kenr Yellow ra, N go -2 -6 10 l5 DI]t (61.20/0) FIG. 4. Discriminant function.analysisof green and yellow morphs of A. gossypii collectedthroughout Lubbock and Brazoscounties, To DISCUSSION The cuticular lipids of most aphids are mainly composedof HCs, but some insects also contain polar cuticular lipids, such as aldehydesthat contain an oxygen functionalgroup on long aliphatic carbonchains (Iockey 1998).In general,the long- chain polar cuticular lipids are waxier and less fluid than the cutioular HCs. Heirce,these t22 protection against lipids function more for lvater impeNmeabilitfaand possibly as profile t inryO"* that has fallen onto the insect cuticle @uckner 1993). In the chernical group' oitrt! "otto" aphidsused for this research,a polar cuticular lipid in the aldehyde l-hexacosanal,was identified, but excludedfrom the analyses' (B)-pfameserre(BBF)hasbeenisolatedandidentifiedasanalarmpheromoneof 1973)' ."uoa "pnla species,inctuaing cotton aphids@owers et al. L972, Edwardset al. and rhis ptreromoneis secretedby-ttre aphid cornicles in responseto attackba pldators pr#ito causingnearby aptri-dsto dirpor" (Wynnand Boudreaux 1972)'EBF was also identifreOin the-chemicalprofiles ofihe cotton aphids used for this research.Although the EBF is not considereda cuticular HC, rather a sesquiterpenecompound derived from ap;a comicte, we included it in the HC analyses.It was found in all profiles in similar aLundancesand so had minimal influenceon the conclusions' Researchon HCs has enrphasizeddifferentiating betwee,nsibling species or al. ,orrrp""in"t that are difficult to identiff on the basis of morphology (Milligan et 1986,Anyanwu et al. 2000). Typically, closely relatedor related samplesshare the same .o.po*it, and differ oniy in-relative concentration(Ryan et al. 1986, Broza et al. iOOlll. ffrut, although sibling speciesor conspecificsmay have the same qualitative pronie, discriminatio-nis basJd on relative concentrationsof the HCs rather than their merepresence or absence(Harnilton and Service 1983,Anyanwu et al. 2000). In this study, we found the alkaneswere identical in both the gree'lrand yellow color morphs for each inatividual population, but the relative concenfiationsof the .o-po*d. differed sigrrificantly. Thesi resultswere similar to the examplesnoted above in that discrimination anrongaphid populationswas basedon relative concentrationsof the HCs rather than their mere presenceor absence.However, a pooled DFA on the samplesfrom all threepopulations revealed sigfrificant differencesacross populations not onty in relative concentrations,but also in the tlpes of HCs. One might expectthat the yeliow and greenmorphs in ttre individual populationswould sharethe sameHCs, but it is not clear wny *t" glographically separatedconspecifics should possessdifferent HCs. This variability in H- piofites within a single specieshas raised questionsregarding the ecological- meaning of this variability(Howard 1993)' Variationsin HC profiles are not linked to the actual chemicalscontributing to give the pigmentationof aphid color morphs.The colored pigments,or _aphins,which rise'to-ttre pigmentation of aphid color morphs occur throughout the hemolymph and body tissues,-but are not foqnd in the aphid cuticle (Wall 1933). The pigme'lrtation respbnsiblefor the coloration of the green and yellow morphs has been documentedto changew ith s easons( SetokuchiI 981).Y ellow morphaphids are m_ostoften found in periods of high temperaturesand crowded conditions. Green morph aphids are more rornlon in lower temperatureconditions where little crowding occurs(Kring 1959). The yellow pignent in the cotton aphid was identified as carotin. Subsequently, the green"p-tiOt *oJfound to possesscarotin in an even Sreat€rquantity. Therefore,it yellow pigment was was-recogpizedthat in 4" gf.* forms, the pfesenceof the carotin maskedb-y the presencebf ttre greenpigment. Thus, any color variationswere due to the differing proportions of green pigment to caf,otin (Wall 1933). Consequently,even though we found that yellow and green morphs had significantly different relative conc-entrationsof HCs, these differencesare not directly associatedwith the chemicals contributing to the pigmentationof the aphids.Althougfu, in most cases,it is not difficult to distinguish gt""" upniat from yellow aphids,we choseto identify the HC profiles of both color morptn. We did not expect to find any differencesin the HC profiles of the two color motphr within the samepopulation, but to our surprisewe did. Fuhre research r23 on cuticular HCs should consider this distinction particularly with respect to pattern recogrition analyses. The characterizationof HC profiles is valuablein the context of taxonomy (Lockey l99l), as demonstratedby the largenumber of HC profiles of variousspecies that have beeninvestigated (Lockey 1991, Singer 1998).Over 400 papersoe cuticularHGs sf arthropodshave been published, but many taxa still need to be characterized(Howard 2001). Profiles are complex, and historically, many compounds were not identified because they were undetectablewith available instrumentation(Inckey 1991). Improvementsin analytical techniquesand insfrumentatiorlhowever, have facilitatedthe isolation and identification of cuticular compounds.We can expect further development of searchprogrums and storageof profiles in data burks which may make routine the identificationofclosely relatedspecies on thebasis ofchemistry. ACKNOWLEDGMENT We thank GeorgeCobb, Yu-Jie Guo and Harlan Thorvilson for help and advice and Richard Straussfor guidanceon statistical analyses.Financial supportwas provided by TexasTech University. LITERATURECITED Anyanwu G. I., D. H Molyneux, and A. Phillips. 2000. Variation in cuticular hydrocarbonsamong strains of the Anophelesgambiae sensustricto by analysis of cuticular hydrocarbonsusing gas liquid chromatogaphy of larvae.Mem. Inst. OswaldoCruz, Rio de Janeiro95:295-300. Bergman,D. K., J. W. Dillwith, R. K. Campbell,and R. D. Eikenbary.1990. Cuticular hydrocarbonsofthe Russianwheat aphid. Southwest. Entomol. 15: 9l-100. Bartelt, R. J., M. T. Armold, A. M. Schaner,and L. L. Jackson.1986. Comparative analysisof cuticular hydrocarbonsinthe Drosophila irilis speciesgroup, Comp. Biochem.Physiol. 838: 731-742. Bowers, W. S., L. R. Nault, R. E. Webb, and S, R. Dutky. 1972. Aphid alann pheromone:isolation, identification, synthesis. Science 17 7 : ll2l -1122' Broza, M., J. L. Nation, K. Milne, and J. Harrison. 2000. Cuticular hydrocarbonsas a tool supportingrecogn:ition of Gryllotalpa tali and G. marismortud(Orthoptera: Gryllotalpidae)as distinct speciesin Israel.Ann. Entomol.Soc. Amer. 93:1022- 1030. Buckner,J.S. 1993.Polarcuticularlipids,pp.22T-270. InD.W. Stanley-Samuelsonand D. R. Nelson [eds.] Insect Lipids: Chemistry, Biochemistry and Biology. Universityof NebraskaPress, Lincoln. Dillwith, J. W., P. A. Neese,and D. L. Brigharn.1993. Lipid biochemistryin aphids,pp. 389-434./z D. W. Stanley-Samuelsonand D. R. Nelson [eds'] hsect Lipids: Chemistry,Biochemistry and Biology. University of NebraskaPress, Lincoln, EdwardsL. J., J. B. Siddall,L. L. Dunham,P. Uden,and C. J. Kislow. 1973.Trns'p- famesene,alarm pheromoneof the greenpeach qhid, Myzuspersicae (Sulzer). Nature241: 126-127. Hamilton,R. J., andM. W. Service.1983. Values of cuticularand intemal hydrocarbons for the identification of larvae of Anophelesgambiae Gies, Anophelesarabiensis Patton,and Anopheles rnelas Theobald. Ann' Trop' Med. Parasitol.77:203-210' r24 HavedyM.I.,L.J'Nelson,andB.T'Forschler'lggg.Newcuticularhydrocarbon- pt rnotl'p es of Reticulitezzes (Isoptera:Rhinotermitidae) from the united states. Sociobiology34t l'21' HebanowskaE., E:-Malfulski, J. Nawrot, M. Ruszkowska,K. Pihlaja' and J. Szafranek' 1989.T he c omposition of c uticular h ydrocarbonso f t he e ered aphrdss inbion avenaeF.Qlomopter4Aphididae).Comp.Biochem.Physiol.94B:723-727. Howard, R. W. 19b3.Cu'ticutarnyArocarbons and chemicalcommunicatiorL pp. 179'226. in D. W. Stanley-samuelsonand D. R. Nelson [eds'] InsectLipit.. Chemistry' Biochemistry .ni eiotogy. university of NebraskaPress, Lincoln, Nebraska. Howard, R. W. iOOt, Cuti.ular hydrocarbons of adrit Pteromalus cerealellae (Hymenoptera: pteromalidae) and two larval hosts, Angoumois grain moth Bruchidae). Ann. iL.piaopt"o, Gelechiidae) and Cowpea weevil (Coleptera: Entomol.Soc. Amet' 94:152-158. Howard, R. w., G. Perez-Lachaud,and J. P. Lachaud.2001. cuticular hydrocarbonsof Kapala sulcifacies(Hymenoptera: Eucharitidae) and its host, the Ponerine ant Eitatommaruidum (Hymenoptera:Formicidae). Ann. Entomol. Soc.Amer. 94: 707-716. Kring, J. B. 1959.The life cycle of the melonaphid, Aphis gossypii Glover, an example of facultativemigration' Arm. Ent' Soc.Amer. 52l.284-286. Leclant, F., and J. P. Degpine. 1994.Aphids (Hemiptera:Aphididae), pp.285'323. In G. A. Mathews and J. P. Tunstall [eds.] krsect Pests of cotton. University Press' Cambridge. Lockey, K. H. i988. Lipids of the insect cuticle: origins, compositionand function. Comp.Biochem. Physiol. 89B: 595-645. I-ockey,K. H. 1991.Insect hydrocarbon classes: implications for chemotaxonomy.Insect Biochem.2l:91-97. Iockey K. H., and N. B. Metcalfe. 1988. cuticular hydrocarbonsof adult Himatisnus speciesand a comparisonwith 2l other speciesof adult Tenebrionidbeetle using multivariateanalysis, Comp. Biochem' Physiol. 9lB: 37l'382. Lockey K. H., aqd V. S. Oraha. 1990. Cuticular lipids of adult Locusta tnigratoia migratoriodes (R and F), Schistocercagregaria @orsk6l) (Acrididae) and other orttropteranspecies - IL Hydrocarbons.comp. Biochem.Physiol. 958].721-744. Milligaq P. J. M.,.4.. ltrillips, D. H. Molyneux,S. K. Subbarao,and G. B. White' 1986' Differentiation of Anopheles anlicifacies Giles @iptera: culicidae) sibling speciesby analysisofcuticular compon€nts.Bull. Entomol. Res.76: 529-537. Ryan L., A. Phillips, P. Milligan, R. Lainson,D. H' Molyneux, and J. J. Shaw.1986' Separation of female Psychodopyguswellcomei and P. Complexus(Diptera: Psychodidae)by cuticularhydrocarbon analysis. Acta Tropica.43: 85-89' Setokuchi, O. 1981. Occurrence and fecundity of two color forms in Aphis gossypii Glover (Homoptera: Aphididae) on Dasheenleaves. Jap. J. Applied Ent. and ZooL 16:.50-52. Singer,T. L. 1998. Roles of hydrocarbonsin the recopition systemsof insects.Amer. Zool.38: 394-405. slosser,J.E.,W.E.Pinchak, andD.R.Rummel.lgg2.Populationdevelopmentand regulationof the cottonaphid, pp. 649-651..InProc. Beltwide Cotton Conf. Vol. II, Nat. Cotton Council of Amer. Munphis, TN' wall, R. E. 1933. A study of color and color-variation in Aphis gossypii Glover. Ann. Entomol.Soc. Amer. 26:425463. wynn, G. G., and H. B. Boudreatx. 1972. Stucture and function of aphid cornicles. Ann. Entomol.Soc. Amer. 65:157-166. 125 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JI.JN.2OO4 SEASoNALPoPULATIONDYNAMICS,LIFESTAGEcoMPoSITIoNoFTHNPS--- NATURAL rlnict wwsAl.topTERA: THRJpIDAE), AlrD PREDACEOUS ENEMIES ON ONIONS IN SOUTHTEXAS Tong-XmLiu TexasAgri""try-ryp"lT.4t vegetable-stutiotu IPM laboratorv Departmentof Entonolory, toras lav university, 24158. High*ay 83, Weslaco,fi 78596839 ABSTRACT Onion thrips, Trrips tabaci Lindernaq is the most important insectPest of onions in the Lower Rio-CranaeVatley (LRGV) of south Texas.The population dynamicsand enerty life stage composition-ooio* of T. tabaci populations and the predaceorrsnatural popdadons on were determinedin tbr€e consecutiveseasons from 2000 to 2002' T.'tabaci were first pr€sqrt on onion plants in ealy February, increald in numbers grO,raty, and peakdin abundancein late March and early April. Vizual countsof field 77o/oof btal predaton by absolde i"e"t"riirl" t"n*f"a 45Yo of tataltbripa and at least loirtt. Developmentalstages of T. tabaci on onion plants consistedof 7685Vo lgttre, {).1% pnpae,-d 1028P/oadults. Although insecticide rylications reduced tbtips far a*Jty,'- ""o"g" of 92 thrips was found on eachonion plant overthe s,Tso9 which o"oiid the ec6nomic threshold. S€v€ral speciesof predatorswere found on onion plants. Orins insidiosus(Say) was the most abundantpredafor species, rryith37.4'74'5o/o bn onion plants, depending on the season and insecticide application' There were sigrificant'conetatioris UetGen predafon and tbrips densitieson rmtneatedonion plants (r-= 0,7327-0.8234),but there were no such correlations on insecticide-beatedonion regulating pt*tt G = 0.053G6.4537).It appearstbat predato'rsw€re not a mqior factor only the early fuoipr bpututions. Of tlre weatb€r conditions, t€tnp€r1lure affected infestatioi in Janury and Febnrary, and a daily rainftll of 1.8 cm or morp caused temporaryreduction oftbrips densitieson onion plants. INTRODUCTION onion (Altint cepa L.) is a major vegetablecrop in south Texaswith >6,000 ha harvestedwith a value of >$80 million in 2001 and an economicimpact of >$150 million (Anonyrnons2001). Onion tbrips, Thrip.rtabaci Lindenan' is one of the most important p""tr ofooio* in south Texas (Royer et al. 1986,Edelson and lvlagaro1988, Edelson et al. t989, Sparkset al. 1998,Liu and Chu 2004). Ntlough conve,lrtional chernical control of T. tabaci on onions is fairly ineffective beca$€ of insecticide resistanceresulting fiom years of otposure to many active ingredients,it is still the only meansof managingthis tbrips on onions in south Texas(Siarks et al. lD8, Liu, unpublisheddafa). Knowledge of the populationdynamics and ff; stageoomposition for a certaintbrips populationin a given field and time may be uscfuf for proviaing manegemelrtrecommendations because different life stagesof tbrips t27 adult-larva G'e', ratio) responddifferently to insecticides(Liu et al., r.rnpublishedclata). For example, population a with more larvae may be more .ur".phtr"'a insecticides comparedwith one with more adults. In contrast,lhrips larvae ;t b" h*d to manage with insecticides becausethey are not migratory and trid'einside the 6;;tir. Natural enemies .of.tbripl especially predarors,may play a significant role in sqpresslng thrips populations (Hotrmurn a a. lDx, sauetis'ana i*-ru;o ter1. Information predatory, ^o! hymenopterouspa*sitoids, parasitic o.utoa", and fungal ry999*r 9f_tt"iprhas been reviewed uy sa*m andvan niio rrsgz),iooo.r* "t ul. (l?97), and Butt and Broumtridge (1997). Howwer, the impo,rtanceofout*I - enemies of thrips, especiallythe pledators,in southrexas hasnot beeninvestigated. The objectives of this study were to detennine:(l) tlre 'rfoi".popuiJon dynamicsand life. stage composition of T. tabaci populations, *b' (z) coilposition of predaceousnatural enemieson onion plants in southTexas. MATERIALS AND METHODS The study was conductedat the ResearchFarm of the Texas A&M university Agriculfural Researchand Extension center at weslaco, Texas. onions (var. "1015'i planted were on a l-m bed and spacedat 25 cm. The plants **r -uiot"in"d under :tt"q{ cultrnal practicesfor south rexas. Each plot naa l0 rows, ana eactrwas 30-m long. The two insecticidesused were, l,-cyhalothrin (warrior with Zeon technology, I EC; Syngenta,Greensboro, Ncl 0.033 kg AI/ha (0.03 lb AVacre), and mettromyl pon! I (LannateL,24yo AI; Du wilningron, DE) at 0.5 kg AI/ha (0.4iib Avacre). Tlie two treatments, an insecticide treatment and an unteated control, each with tbree replications,were anangedin a randomizedcomplete block design.soighum was planted algng the margins of the plots as a windbreak to reduce por*itte ui*e*-ptoi ttrip, migration. Herbicides (bensulide [prrefar4E], Gowan, yrma, Az; l,ll2 g enal *o fungicide (chlorothalonil [Equu 720], criffin, valaostq GA; 1,260 geLntut-*o" appriea as needed. Utt! pt*t sampling methodswere used in 2000. Thrips sampling beganon 7 February 2000 and carried out weekly until harvest. when sampling, to-onio-n ptants randomly selectedfiom eachplot were cut at ground level beforettt" uutu *as f*.d, o, were cut ftom the neck or the upperpart of the bulb after the bulb was formed.The plants yog in{ilaualy placed in l-gallon ziplock plastic bags (s.c. Johnson& son, Inc., Racine, w). In the laboratory the leaves of the plant wele s€paratedinside the plastic bag and rinsed with water. All tbrips, natural e,nemies,and larvae inside the basesof onion leavesor near the neck were washedoffwith a bottle sprayer.All ins€ctswashed o^ffeach plant wgre filtered in a firnnel, and wenetransf€rred to a clear plastic petri dish (9'cm diameter. xil.S-cm deep). Thrips adults, larvae: pupo€, and predaton were identified and counted.visual counts methodswere used fuf2001 and2w2. From early February,10 onion plantswere tandomly selected,and numbersof adult ttnips and larvae were visually counted in the fields. All voucher specimenswere depositedln the Insect collection of the TexasA&M University Agricultural ExperimentStation at weslaco. The weathet data were obtained from a weather station monitoring temp€ratrf,e, rainfall, and wind direction and speed located approximately IOO; from the experimentalonion field. Dafa on thips collected fiom onion plants, blue and white plastic cup taps-lvteans and cc krys were analyzedusing analysisof variance(AlrovA, sAS Institute2obz). yere_s€parqed using the honestly significant difference t€st or T*€y test after a significant F-test at P = 0.05 (7ar 1999). Becauseonly a few pupaewere collectedfiom t28 larvaein the onion plants,representing <1% if the total thrips,they were combinedwith predatorswere ;;G;;.-a;""lations oI the numbersof thrips with the numbersof analyzedusing PROC CORR (SAS krstitute 2002)' RESULTSAND DISCUSSION Becausethere were only a few F. occidentalis(<17") found in the onion field (Liu and Sparks, unpublislred data;, they were not separatedin dafa analysis in the thrce t""ro*. tluipsiopulation abundanceon onion plantsfluctuated though the seasonin all tlree seasons(nig. t). Generally,thrips were pr€senton onion plants in early February, and increasedin abrmdancegradually to the end of the seasonin mid or late April. 3 9zo E E ! 28 815 .E g o E c - T€mperatjG 810 : Rahlbll ;I + Ao.llttlbit + Flrc.Llnrlattd u c, g3m e e 2oo ts ?r'1totr 3r2roo 41rn 2 n1 3,30' 1 1t201 5t2h1 211M2 38102 lnNt g2n2 I)!b (rntuy) FIG. 1. Populationdynamics of Thrips tabaci or onions and ternperatureand rainfall in the springsof 2000 to 2002 (Weslaco,Toos). Thrips populationson onion plants as determinedfiom both the absolutecounts and field visual countswer€ motreabundant tbroughout the 2000 seasonrelative to those in 2001 nd2002 sea$tns.In the 2000 seaso&thrips abundanceaveraged 222 nd 116 thrips per plant in rmteated and insecticide-tneatedplots by absolutecounts, respectively, and 167 and 91 thips per plant in rurreated and insecticide-trededplots by field visual counts, respectively 6able l). Thrips densitiesalso varied greatly arnongthe plants as indicated by the extsemelywide rangps,from 2 to 979 thrips per plant. Although tbrips populationson rmteated plants in 2001 and 2002 were lower than thosein 2000, average numbersofthrips over the two seasonswere still as greatas 102 and 92 per plant in each ofthe two seasons,respectively. 129 TABLE l. Mean Abundance of rhrips tabaci per onlon plant and Adurts/Larval Compositionon OnionPlants (Weslaco. Texas). Seasonandheafuelrf 2000:AC, 116.4+7.2 .l + 1.7 27.6* l.t 5.0+ 0.4 2000: AC, unteated 221.6+tt.t 34.8+1.9 19.0+0.7 6.7+0.3 2000: FVC, treated 91.3+8.9 16.2+3.1 t7.7+2.8 5.6+0.9 2000: FVC, unbeated 166.8+21.826.4+3.1 15.8+1.2 5.3+0.8 2001:FVC, rmtneated 102.1+ 7.5 7.3+ 0.6 10.4+ 0.8 2t.l + t.9 2002:FVC, rmtneated 91.9+ 4.9 9.9+ 0.5 18.0+ 1.0 16.4+ 1.6 B. 2mO Field visual @unts + On untreat€d plants o1ll ;0 t 2n/i/002t21t@ 3/6/003/20,00 4r3/IJf) 417rn 2nno 2r21m 316100320,t)0 4t3t@ 4t17M Eso s C. 2001Field visual @unts 2002Fleld visual counts On untr€aledplanls On unfeatedplants 0r- t5/0t u19/01 4/2n1 4n6n1 4A001 3t4to2 snEn2 4t1t02 4t1WI2 4ti2g02 Dato(n/dfy) FIG. 2. Percentagesof adultThrips tabaci ononionplants sampledusing absolutecounts and field visual countsin the springsof 2fiD to2002 (Weslaco,Texas). Applications of insecticides(Lcybalothrin and methomyl) in 2000 significantly r€duc€dtbrips populationson onions comparedwith untreatedplants basedon absolute counts(F: 14.96; df = 1, 580; P < 0.0001) and field visual counts(F = 151.27;df : l, 580; P < 0.0001). However, three applicationsof l,-cyhalotlnin and five applicationsof methomyl did not effectively suppressthrips populations on onions during the seasor! 130 and nlmbers of thrips on insecticide-tFeatedplants were still greaterthan thd economic thresholdof I thrips per plant @delsonet al. 1986). M zno Abooltfrecouils Fiddvbudc.|rils + orrheeteddantB + orrteateddanb + Onunbealedddtb + Orutr&dddtts 2 uJ !,r E .g e0'x7n0 e TAI@ 36rm 3fiXlm 4nm 4flt6 2I7n 2D1t6 3,hit0 SAym *?tt@ $fltn Bto E 20D1 2W a Fieldvi.ual cout3 Fiddvialel cour0s zoB 6 0]- 2t5t012/1E013/5/01 3t't9/0il +?JOI .lrlc,ol 4J:m1 Z&22t1882,31ffi,311l3,0n41&24/16//@A1nff2 tlafie(rJd/y) FIG. 3. Predalors of Tlrips tabwi on onion plants in the springs of 2000 ta 2002 (Weslaco,Texas). Weather conditions could sigtificantly afrect the population dynarrics of Z tabaci on onions (Fig. 1). Of the environmentalconditions, heavy rainfall ruasthe most important factor regulding thrips densitiesin this orperiment (Figs. l, 3). For example,a rainfall of 3.7 cm with a 51.5 kdh (32 mph) wind on 14 March 2fi)0 was followed by a reduction inT. tabaci densities,especially lawal thrips, on onion plants.The rainfall was followed by thrips population rapidly incrcasing for alnost three weeks. In 2001, a rainfrll of 1.83 cm on 3 Mach was not followed by a reduction in thrips poeutations becausethrips populatioilr wer€ already low, but a rainftll of 1.80 cm on 24 April was followed by a rcduction in ttuips populationsfollowing rain wents s/ l3r plants and 19.0%oon untreatedplants in 2000, to as low as lo.4% n zo0l .(Table l). Generally, adult thrips composedof 4v/o of all thrips on onion plants as estimatecty both the absolute counts and the field visual counts. The high€r percentag€sof aduit thrips to total thrips werc found in the early seasonwlren thrips infestationwas at an eaily phase (Fig. 2). with the incftase of adult thrips populatioir, mofe imrnatures were produced, lowering the adult percentage.The lofver p€rc€ntagesof adults in the late seasoncould be causedby fewer luvae molting to adults, or more adults leaving the plants as the plants becamematwe. In additioq adult tluips may migrate from plants, especialll when plants were senescing.tn the fields where insecticideswere applied,the insecticidesmight initate the adult thrips and causemigration to adjacenturtneated plants or wild hosts. Several species of predators were found on onion plants, includng Orius insidiosus (Say), bigeyed bug, Geocorispunctipes (Say), assassinbug, Sinea sptnipes (Herrich-schaeffer), chrysoperla rulElabris@urmeister), several species of lady beetles, sprders,predacious mites, artd rove beetles. Most predators found in this study are generalistpredators. Generally, the nunbers ofpredators were extremely low on either insecticide-teatedplants or untneatedplants in the eady and mid-seasoo,and peakedin the late seasonin April when thrips populationspeaked (Fig. a). Numbers of tbrips and numbers of predators on wrtreated onion plants were correlated" with correlation coefficients rangmg from 0.73 to 0.82 (Table 2). However, numbers of thrips and numbersof predatorson insecticide-treatedonion plants were not correlated(r : 0.05 to 0.45). TABLE2. CorrelationofNumbersof Tlrips rabaciandNumbersofPredatorsonOnion Seasonand neatnent 2000:absolute counts 0.0536 0.759f 2000: field visual couts o.4537 0.8234b 2001:field visualcounts 0.7327^ 2002: field visual counts 0.7&g' ", " Significant at P = 0.05 and 0.01, respectively(Tukey test, SAS Institure2002). TABLE 3. Nnmbersof PredaceousArttuopods of Thrips tabaci per Plant and hoportion of Orirs ^Insidlbsz,son Insecticide-Treatedor UntreatedOnion Plants(Weslaco, T)O Treated Unheated seasons" ffi ffi F 2000Ac 0.20+ 0.03 40.2 1.87+ 0.13 74.5 22t.890 2000FVc 0.24+ 0.03 43.1 1.44+ 0.11 72.3 13638b 2001Fvc 1.69* 0.19 37.4 2002FVC 1.26+0.32 42.7 " AC: absolutecouts; FVC: field visual counts. b Sigdficant at P = 0.01 (Tukey test SAS Institute 2002). Ofthe predatorsfrom the absolutecounts in 2000 or field visual counts in the thtee seasons,a majority were Orius insidiosas(Say) (Table 3), rangng from65.2-74.50/o of total predators.However, on insecticide-tneatedplanrc in 2000, only 40.2 atfr 45.2o/o were O. insidiosus, which were sipificantly different from percentageson unteatpd r32 onion plants. Numbers of thrips per Orius ranged from 23 in eatly Februaryto 177 n early April on untreatedplants and 13 in early Februaryto 458 in early April on insecticide-teatedplantsin2000,from25to171 thripsperOrius in200l,andfrom l5to 273 in2002.It appearsthat predatorswere susceptibleto insecticidesas shown from the data in 2000, and that numbersofpredators were significantly greateron unteated plants thanon insecticide-featedplants in both absolutecounts (F=221.89; dFl,580; P<0.0001) andfield visualcounts (F=221.89; dFl,580; P<0.0001)(Table 3). DISCUSSION Onions are normally directly seededin October, but can be planted early in Septemberto late in November or Decernberin south Texas. After germinating 5-7 d after seeding onion seedlingsgrow slowly during tb€ wintsr and are normally in the 5-7 leafstage by January.Although the first presenceofonion thrips on onion plants depends on the temperaturein the early spring, the cool weatherduring Decernber,January and Febnrary,rvtich approximatesthe lower dwelopment threshold(l 1.5"C) for T. tabaci, is the causeof low populations dring these months @delsonand Magaro 1988). Thrips infestationsin fields strt to inq€ase as early as Jmury, but normally in eady February. Populations increasc gra&ully with the go$ah of onion plants, and rcach peaks in abundancein late March or early April; however, there is no significant conelation betrreent€mp€rafrr€ and thips densrty.Despit€ the substantialdifferences in the thrips popnhions dudng tbe thee seasonsrn 20/JJ.20f.ll2,onion tbrips population dynmics patt€rn anong the tbree consecutiveseasoilr coincided with the nomral patt€rn of thips occurr€nceon onion plantsunde,r field conditionsin southTer6s. The hot, dry conditions in south Toras in the springnot only favor the onion crop growth but also thrips population increases.As shorm in Fig. l, rain is rare in the spring in south Terras.In tb€ tbr€e seosons,only a few days received a rainfall over I cm. Therefore,rve do not normally e:rpectheavy rainfall to occur and washtbrips from plants. Although t€mperatureand ttuips densrty is not conelal€d over the sea$rL the low t€mp€raturesin Januaryand Februaryin 2001 nd2002 might preventthips population fiom rapid incrcasinguntil mid-Much (Fig. l). In conhast teinperatrnesrcached 2(PC or higb€r in early Februaryin 2000, and the thrips populationsincreased rapidly from mid- Februaryand peakedin early lvladr Theseresults indicate that temperaturein the early spring can play a significant role for early seasontbrips infestation and populaion increase. R€sults fiom tbe 2fi)0 seasonshow that applications of insecticidesrduced predaciousnattual enerny abrmdance.For instance,only 40.2 and,43,1o/o of predators wene O. insidiosus,which is significantly different Aom untreatedonion plants. These low perceirtagesof Oius to total pedators in the insecticide-treatedplots indicafe that Ortus mi$t be more suscepdibleto insecticides than otber preaafn. These results confirmed that @acious insectsare tpically mo'rezusceptible to insecticidesthan the phytophagouspest spociesdue to the evolution ofa mechanismfor detoxificationofplant secondarycompourds (Croft 190). Natural enemies, inclding preddon, hymenopteran parasitoids, parasitic n€Nnatd€sand fuSal pathogensof tbripc can play prominent roles in regulating thrips populations on plants under natural conditions (Hofuann et al. 1996, Sabelisand van Rijn 1997, toomans et al. 1997, Butt and Brownbridge 1997). There is no doubt that natural enemiescan be successfullyus€d to utaoagethrips in grcenhouses(Jacobson 1997). However, therp are coffioversies regardingthe importmce of natural enemiesin suppressionof tbrips populations in the ficld. Panella and Lewis (197) indicafod that 133 natural enemiesplav an insignificantrole in rezulatingthrips populationsunder field conditions. Although t.heresults in this study indicate that predrationby natural enernies was not a major factor or was at least not adequatein suppressingthrips populdions on onions, it is difficult to make firm conclwions about the impact of nanral enemiesin a field'n}ere the thrips populationexceeded an eoonomicthr€sholdbysuch a large margin. In addition, these predatorsmay be hamperedby the fact that thrips feed under close- fitting leavesand down in the leaf sheathswhere they are difficult to access.Also, when thrips populationswere as high as thosein 2000 and 2001, it may maskthe role of natural enemies. ln conclusion, T. tabaci is the most important pest insect affecting onion quality and yield in south Texas.Field visual countsestimated 45o/o of niarl thrips and 77D/oof total predatorsby absolutecounts. of the developmentalstages of r. tabaci on onion plants, 76-85% were larae, <0.1olowere pupae,and l0-2}o/owere adults.Application of insecticidessigrificantly reducedthrips densities,but therewere still >90 rhrips per onion plant throughoutthe season.several speciesofpredators were found on onion plants,and a majority of thesepredators were o. insidiosw. Temperaturesin Januaryand February affect early thrips infestations,but have no significant effects thereafter.Heavy rainfalls can temporarily wash offthrips densities,but it is rare in southTexas. Although the level of infestation by T. tabaci can be extremely heavy during March and April before hawest, at presentthere are no effective managementmerutures (Spmks et al. 1998, Liu and Sparksunpublished data). Becausethere are no thrips-resistantvarieties @delsonet d. 1991, Hamilton et al. 1999), an integrated thrips manageurentprognrn, including monitoring thrips infestation levels, reevaluatingcurr€nt economic tbreshold, s&ae€ning efficaciousinsecticides, and determiningthe potential of biological contol, is needed. ACKNOWLEDGMENT I greatly appreciate Drs. Jonathan V. Edelson @epartment of Entomology, Oklahoma State University, Lane, OK) and Noel Troxclairs (Texas Cooperative Extensioq Uvalde) for reviewing the manuscript,and J. Martinez, M. I. Moral, and M. De Leon for technicalassistance. Publication ofthis manuscripthas been approved by the Director of the Texas Agriculttral Experiment Station at Weslaco,and the Head of the Deparfinentof Entomology,Texas A&M University, CollegeStation, Texas. LITERATURECITED Anonymous.2001. 2000 TexasAgricultural -Statistics,Texas Departrnent of Agriculture Bulletin 258, TexasAgriculural Statrttics Senrice,Austin, TX. Butt T. M., and M. Brownbridge.1997. Fringal pathogens of thrips, pp.399433. InT. Lewis [ed.]. Thrips as crop pests.CAB, Wallingford, Oxon, UK. Croft, B. A. 1990.Arttropod biological conhol ag€Nrtsandpesticides. Jobn Wiley & Sons, New York. 723 p. Edelson. J. V., B. Caffu/right, and T. Royer. 1986. Distibution and impact of Tlvips raDaci(Thysanoptera: Thripidae) on onion. J. Econ. Entomol. 79:5A-505. Edelso4 J. V., B. Cartwrighr, and T. A. Royer. 1989. Economicsof conEolling onion thrips (Thysanoptera:Ttuipidae) on onions with insecticidesin south Texas. J. Econ. Entomol. 82: 561-564. Edelson. J. V., and J. J. lvtagaro. 1988. Development of onion thips, Tlrips tabaci Lindeman (Thysanoptera:Tbripidae), as a firnction of temperature.Southwest. Entomol.13:71-176. 134 Edelson,J.v., J.J. Magaro,and T.A. Royer. 1991. onion cultivar yield responseto onion thrips intbstation,pp. l-3. TexasAgricultural ExperimentStatioq ltogress Report,PR-4815, College Statio4 Texas. Hamilton,B. K. L. M. Pike,A. N' Sparks,Jr., D. A. Bender,R. W. Jones,J. Candei4and 'IPA-3' G. de Franca.1999. Heritatiility of ttuips resistancein the onion cultivar in SouthTexas.Euphytica 109: ll7 - 122. Hoftnann, M. P., C. H. Petzoldt and A. c. Frodsham.1996. Integml€d Pest Management For Onions. Cornell University, Comell, NY. Jac,obson,R. J. 1997.Integrated pest management(IPM) in glasshouses,pp.639'666. In T. kwis [ed.]. Tbrips as crop pests. CAB, Walingford, Oxon"UK. Liu T.-X., and C. C. Chu. 2004. Comparisonof absolute estimatesof Thrips tabaci (Thysanoptera:Ttnipidae) with field visual counting and sticky faps in onion fields in south Texas.Southwest. Entomol. This issue. Loomans,A. J. M., T. Muai, and I. D. Greene.1997. Interactionswith hynenopterous parasitoidsand parasiticnematodes, pp. 355-397.In T. Lewis [ed.]. Thrips as crop pests.CAB, Wallingford, Oxon, UK. Parrella,M. P., and T. Lewis. 1997.Integrated pest managernent(IPM) in field crops,pp. 595-614. /z T. Lewis [ed.]. Ttuips as crop pests.CAB, \trallingford, Oxon' UK. Royer, T.A., J. V. Edelson, and B. Cartunight. 1986. Damage and control of Thrips tabaciLindemra on spring onions. J. Rio GrandeValley Hort. Soc.39: 69-74. SASInstitute. 2002. SAS/STATusers' guide, Version 8.01, Cary, NC. Sabelis,M. W., and P. C. J. van Rijn. 1997.Predation by insectsand mites, pp.259-354. /z T. Lewis [ed.]. Thrips as crop pests.CAB, Wdlingford, Oxon" UK. Sparks,A.N. Jr., J. Anciso, D. J. Riley, and C. Chambers.1998. Insecticidal contol of thrips on onions in south Texas: Insecticide selection and application methodology.Subtrop Plant Sci. 50: 58-62. Zat,J.H.l999. Biostatistisalanalysis,4e Edition. Prentice-Hall, Englewood Cliffs,NJ. 135 voL.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 EFFECTSoFKAoLINPARTICLEFILMoNSELECTEDARTHR0PoD OF TEXAS POPULATIONS IN COTTON IN THE LOWER RIO GRANDE VALLEY Allan T. Showle# and MamoudouSdtamouz ABSTRACT Lraf cormts and dvac sampling indicated that cotton aplnd, Aphis gossypii Glover, populationsincreased in kaolin-treatedcotton, Gossypiumhirsutun L., plols comparedto Luofn-fr"" control plots, but cicadellid populations were suppressed. Populations of dipterans,Orius spp.,andwasps were reducedin the kaolin treatmentsonly on one of l0 sampling dates over two seasons(2000, 2001). Foliar kaolin sprayshad no effect on other arthropod groups identified in this study (silverleaf whitefly, Bemisia argentifolii Bellows aod Perring; herbivorous hemipteransand coleopterans;thrips; lepidopteran larvae; Geocoris spp.; NaDis spp.; reduviids; coccinellids; Collops spp.; neuropterans; andspiders). INTRODUCTION Kaolin is a white, porous,nonswelling, non-abrasive fine grainedplaty aluminosilicate mineral (AlqSLOro(OH)s)that dispenesin water and is chemically inert over a wide pH range(Harben 1995). Coating gradekaolin is > 90% pure and has a brightnessqualtty of > 85% (Harben1995). Iqiury to somecrops causedby insectsand pathogenscan be reducedby coating plants with kaolin (Glenn et al. 1999). The film makesthe host plant visually or tactually unrecognizable,and particlesadhering to the arthropod'sbody might impedemovement and feeding. Applicationofkaolin padicle film to orchardcrops has resulted in the suppressionof injurycausedbypearpsyll4 CacopsyllapyricolaFoerster; spireaaphid, Aphis spireacola Patch;potato leafrropper,Empoascafabae (Hanis); codling moth, Cydia pomonella (L.); obliquebandedleafrollet, Choristoneura losaceana (Harris); root weevil, Diaprepei; abbreviatus (L); and twospotted spider mite, Tetranychusurticae Koch (Glenn et al. I 999, Knight et al. 20[,0, Lapointe2000, Puterkaet al. 2000, Unruh et al. 2000). Ikolin, applied weekly and biweekly, has been shown to deter adult boll weevils, Anthonornus grandis grandis Boheman,from ovipositing on cotton, Gossypiumhirsutilm L., squares in the Lower Rio GrandeValley of Texas(Showler 2002a);however, its eflects on other herbivoresof cotton, as well as on nontaf,getinsects, are not known. This study was undertakento examineeffects ofkaolin particle film on other herbivorousarthropods and naturalenemies cornmon to cotton fields in the lower Rio GrandeValley. MATERIALS AND METHODS The kaolin used in this study was 'Surroundil' wettable powder (Engelhard,Iselin, NJ) processedto a bright white color of >85o/o,32ym.particle diameter, and coated with a vusoe-aRssARc, weslaco, TX ?Texas A&M University, Weslaco,TX t37 proprietarysynthetic hydrocarbon to impart hydrophobicquality. _ Twenty-fourplots, each 8.lm wide (8 rows,row spacing= lm) x 15.2mlong (0.0125- ha) -witfr 1 l_-m bare ground buffer between plots were aranged in a completely randomized design at the USDA-ARS Kika de la Guza subtopical egriiunua Researchcenter, weslaco, Texas. cotton (var. Deltapine-50)was planted on 6 March 2000.and on 12 March 2001. The herbicidependimethalin (prowlN 3.3 Ec, American pvana$0, Parsippann NJ) to confrol weeds at 9249 a.i./ha was applied by tactor imrnediately after planting. weed control was thereafter conducted-with a rolling gultiyator and by.hand+oguing. Inigation occurred at the start of bloom (mid-Mayi Beginningon ll April 2000and 17 April 2001,when cotton plants had reachldpinhead squarestag€i kaolin suspensionswere applied with a tactor-mbunted boom sprayerusing 18 Teejet 8003E nozzleslm apart (eachnozzle ^, 30cm directly over the top ofa row) at 42.3 liters/haat a pressureof 3.5kg/cm2.Treatments were reappliedweekly-to eight piots and fortnightly to eight plots until 2l June 2000 and 25 Jud 2001. Each apptication consistedof two passesby the tractor to maximize coverage. Three weeks after ttre fint applicationeach year, two 47-cm drop nozzlesper row were addedto increasecoverage. The remaining eight plots were not teated (kaoiin-free). No insecticideswere appliedto anyof theplots. K-aolin particle- retention on cotton leaves at 4h, lwk, and 2wk after the first application in 2000 was measuredon randomly selectedfully expandedleaves collected fom-the biweekly teated plots. The kaolin was washediom'the leaves (kaolin was forud on both sidesof the leaves)with methanolinto pre-weighedplastic distresusing a 6-mm flat ox hair paint brush to dislodge particles ttiat uaU"a to the leaf and cotton squaresurfaces. The ..9Tol was evaporated,and the dried particles plus the plastic dish were weighed. The difference betrvien initial and final wei'ghtsyierieJ tne massof kaolin on eachleaf. Total ieaf areawas estimatedas two times the measuredleaf areaof one surface using a Model 3100 Area Meter (Li-cor, Lincoln, NB). The massof kaolin collected. from eachleaf was divided by total ieaf surfacearea to giue the massof kaolin oeposttedper cm'. A randomly -. selected leaf from among the top six fully expanded leaves on 50 different cotton plants was-examined in eachplot foi cotton aihids'on 2l April, 5 and 19 May,and2June2000;and.o12. lAgril 2l May,and4June2b0t. otherartluopods,and cotton aphids, were sampled by placing a (The Dvac Company, V"ntur." Ce) orifice. (33-cm {vac diarn) directly onto cotton foliage at five random to"ations on the four of :"ntrl^Tyr 91h plor Dyc samplingwas cinducted forhightlt fro;-Zi eprit to Z June2000, and 23 April to 4 June20-01. Insectscaught in the aiac colteciioi uug, *"r. taken immediately to the laboratory where they wire placed in ja.s containing zox ethanol, identified, and counted. Numbersof cotton plants per 4m;f ro; ;;re counted on 5 and9 July in 2000and 2001, respectively (z=g). Repeated meas*res analysis was used io detect significant differences between teatl^lents -and_sampling dates, and interactions. Insect numbe^ were log(x+l)- nansforme! before repeated measuresanalyses; however, untransformJ means are presented(Analytical Software I 99g). RESULTS Meanparticle_density . on_leaves4h afterapplication was 360.0+1g.7 pg kaolin per cm2 leaf surface. After I and 2 weeksin the fieid, particledensities *"r"its.9*20.g and 201 .0+l 3.2 Fd cm2,respectively. Repeatedm€asures analyr! on_-numbersof aphids found on reavesin eaoh year detected teatment (dts-2, 84) effects (2000, i=6.4g, p=0.0024; zooi-, r=qa.u, 138 P<0.0001),time (dF3, 84) effects (2000, F=155.21,P<0.0001; 2001, F=545.94, P<0.0001), and an interaction between heafrnents and time effects only in 2001 (F13.65, P<0.0001) for numbersof cotton aphidsper leaf. Kaolin effects were highest in the weekly kaolin ts€atnent in late April of both years when populationswere also high (Fig. l). Bv early May, populationshad declined bv n.6,0%,which explains the significant time effect and on the last two samplingdates, mean aphid populationswere t6 * Gontrol2000 ,-14 -O- Control2001 G + Kaolin-weekly2000 o - - ;12 w- Kaolln weekly 2001 o +- Kaolin - blweekly2000 -€- Kaolln-biweekly2001 o- 10 E .tr9 EL o cto C c4 (E o =2 41214t23 515517 51195t21 612614 Date FIG. L Mean numben of cotton aphids(!.SE) per leaf (selectedfrom the upper six tully expandedleaves, n=400), Hidalgo County, Texas. Herbivorous artlropods counted in dvao samples during 2000 and 2001 included aphids, silverleaf whit€flies (Bemisia argentifolii Bellows and pening), cicadellids, herbivorous hemipterans(mostly mirids, coreids, and pentatomids) and coleopterans (mostly elaterids and anthicids), dipterans (mostly drosophilids, cecidomyiidi, and muscids),thrigs, and lepidopteranlarvae. Natlual enemieswere comprisedof Geocoris spp. (Lygaeidae), Orius spp. (Anthocoridati), NaDu spp. lNabidae;, reduviids, goccineflids, collops spp. (Melyndae), neuropterans (mostly chrysopids and hemerobiids), wasps (mostly braconids, eupelmids, etrrytomias, ichneunronids, gteromalids,sphecids, and nichogrammatids),and spiders(mostly clubionids, linyphiids, lycosids,salticids, and thomisids). Kaolin sprays significanfly decreasedmean numbersof cicadellids and dipteransin 2000; in 2001, kaolin significantly increasedaphids, but reduced mean numbers of 139 cicadellids, dipterans, Orius spp., and wasps (Table l). The effects of time were significantwith 16 and 15 ofthe l8 arthropodgroups in 2000 and 2001, respectively (Table l). Only Collops beetleswere not affectedover time, but meanpopulations were consistently <0.5 beetles per dvac sample. Interactions between treatment and time effects were detected for dvac collected aphid populations in 2000, and for aphids, dipterans,Orius spp.,and coccinellids in 2001(Table l). Dvac collected cotton aphid populations(Fig. 2A') in 2000 were highest in both of the kaolin heatmentsin early May when populationsin the control plots declinedmore than populations in tle kaolin teatments. By the third sampling date in mid May, cotton aphid populationsin the control had nearly disappearedbut higher populationspersisted in the kaolin teated plots, particularly those that were treatedweekly, until early June. During 2001, dvac collected cotton aphid populationswere higher in both kaolin treatrnentsthan in the control, but averagepopulations were relatively low (< 30 aphids per five suctions)throughout the samplingperiod. Cicadellid populations(Fig. 28) during 2000 were suppressedby kaolin treatmentson the last tfuee sampling datesas comparedto the control, but in mid-May and mid-June 2001, populationswere lower in the weekly kaolin treatedcotton than in the control. Cicadellid populations in 2000 significantly increased until early June, after which averagenumbers tended to level off until the last sampling date in mid-June. During 2001, cicadellidpopulations in the control were substantialon the first, third, and fifth samplingdates, which causedthe significanttime effect. TABLE l. Comparisons,Using RepeatedMeasures Analyses, of Numbersof Selected Herbivorous and Natural Enemy Arth,ropod Groups Collected by Dvac from Cotton Plantsin Kaolin-Treaterland Kaolin-Free Small Plots. Hidaleo Co.. TX.2000 and2001. Artluopod Eflecg Year groups Treatnent 2000 Cicadellidae 14.7 2,105 < 0.0001 Dipter# 8.9 0.0003 Time Aphidida# . 29.6 4,105 <0.0001 Aleyrodidaes 105.5 < 0.0001 Cicadellidae 34.5 < 0.0001 Herbiv. Hemipter# s8.7 < 0.0001 Herbiv. Coleopteras t0.l < 0.0001 Dipteras 8.8 < 0.0001 Thripidae 54.4 < 0.0001 Lepidopteralarvae 5.1 0.0009 Geocorisspp.a 22.2 < 0.0001 Orius spp.L 64.1 < 0,0001 Reduviidae 4.4 0.0024 Coccinellidae 18.1 < 0.0001 Formicida# 6.2 0.0002 Waspsv 10.6 < 0.0001 Neuropteras 4.6 0.0020 Spidersry ).5 0.0131 Interaction Aphididaea 29.6 8, 105 < 0.0001 140 TABLE 1. Continued Treaturent 2001 Aphididae4 4.1 2, 105 0.0198 Cicadellidae 5.8 0.0041 Dipterac .. 9.7 0.0001 Orins spp.tr 5.8 0.0042 Waspds 4.2 0.0180 Time Aphidida# 88.8 4,105 <0.0001 Aleyrodidaes 60.2 < 0.0001 Cicadellidae 5.2 0.0007 Herbiv. Hemipter# 36.0 < 0.0001 Herbiv.Coleopteras 12.3 < 0.0001 Dipteras 35.0 < 0.0001 Thripidae 38.9 < 0.0001 Lepidopteralarvae 5.5 0.0004 Orius spp.L. 54.4 < 0.0001 lVaDisspp.s 5.3 0.0006 Coccinellidae 23.6 < 0.0001 Formicidal 4.7 0.0015 waspss 15.6 < 0.0001 Newopter# 9.3 < 0.0001 Spidenlu 18.2 < 0.0001 Interaction Aphidida# 2.0 8,105 0.0500 Dipteras .. 4.3 0.0002 Orius spp.L 2.5 0.0171 Coccinellidae 3.9 0.0004 samples(five suctionsper sample,z=8, one d-vac suction is 0.8m2)were taken at 2-wk intervalsfrom 211 April to 16 June2000 and from 23 April to l8 June2001, interaction, treatment*time. z=8, one d-vac suctionis 0.8m2. uOnly arttropod groupswittr sigrificant (P < 0.05) effectsor interactions presented. g.Mostly are Drosophilidae,Cecidomyiidae, and Muscidae. s Cotton aphrd,Aphis gossypii Glover. s Silverleafwhitefly. Bemisiaarsentifolii Bellows and Penins. s Herbivorous Hemipteranswas mostly comprised of Miridae, with lesser numbers of Coreidaeand Pentatomidae. s HerbivorousColeoptera was most comprisedof Elateridaeand Anthicidae. ryFamily Lygaeidae. sFanily Anthocoridae. v. At t a spp. andS o l enops i s gem i nota. s.Mostly Mostly Braconidae,Eupelmidae, Ewytomidae, Ichneumonidae, Pteromalidae,Sphecidae, and Trichogrammatidae. q Families Chrysopidaeand Hemerobiidae. ry Mostly Clubionidae,Linyphiidae, Lycosidae,Salticidae, and Thomisidae. vFamilyNabidae. Dipteranswere more abundantin the contol than in either kaolin treatmentearly in the season(Fig. 2C) during 2000. In 2001, dipteran populations in the conhol were l4l /t -+ Control2000 ,4. F O- control2ool 200 Kaolln. woekly2000 o1o o V- Kaolln - weekly p p Kaolin- blwsokly2000 t l- Kaolln-blwsskly E8 F. 150 t C' .96(, c 1oo g d4 o tr o // L =50 F2 o ,1,--t , Eo )=6rl-S:-iql- 40 15 o d. tr CL o o bso o CL .= 10 E o d20 c d tr tr5 IE tr o10 o o = =o 0 60 4t214t23 3t53n $19521 6/2614 6fi66fi8 Date g. 50 o 5oo dto tr Ezo = 10 4t2t 4t23 5t5 5n 5119il21 6t2 AI4 6t166t18 Date FIG. 2. Mean numbersof selectedarthropod groups (! SE) collectedfrom cottonplants by dvac,Hidalgo County, Texas; A, cottonaphids: B, cicadellids;C, dipterans,D, Orius spp;E, wasps. higher than at least one of the kaolin heatnents in late April, mid-May, and early June, although differences between the populations were less pronouncedthan in 2000. A treatnent*time interactionwas detectedfor dipteranpopulations in 2001 (Table l). Orius spp.populations in 2001 were greaterin the weekly kaolin treatmentthan in the biweekly kaolin treatmentand the control (Fig. 2D). A treatrnent*timeinteraction was detectedfor Orius spp.populations in 2000 (Table 1). 142 Waspswere more abundantin the control than in either of the kaolin treatrnentson 23 April 2001, but populations were relatively low (<5) comparedto the other sampling datesin 2000 and 2001 (Fig. 2E). The effects of time on the other insect groupsin Table I were causedby population increases,particularly from early seasonlows to mid or late seasonhighs. DISCUSSION Applications of particulate materials to crops can adversely affect some pest arthropodswhen abrasionof the cuticle or stuctural disruption of the epicuticle induces waterloss and desiccation(Kalmus 1944, Hunt 1947,Daid and Gardiner1950, Ebeling and Wagner 1959). The spottedcucumber b*tle, Diabrotica undecimpunctatahowardi Barber @ichardson and Glover 1932), and the walnut husk fly, Rhagioletis completa Cresson(Boyce 1932),ingested particles that causedmortality by pluggingthe hindgut. Particlesthat cling to artlropods' bodies can causethe arthropodsto leave the plant as hasbeen suggested for pear psylla, potato leafhopper,and root weevil (Glenn et al. 1999, Lapointe 2000). White reflective surfaceshave been shown to repel some insects by making the plant less visually recognizable(Kennedy et al. 1961,Kring 1962). Showler Q002a) showedthat foliar spraysof kaolin deterredfeeding and oviposition on cotton, and it was suggestedthat preferencewas basedon visual cues. Although spirea aphid populationswere lower on kaolin-treatedapple leavesthan on controls (Glenn et al.1999), the greatercotton aphid abundanceson ootton sprayedwith kaolin might have resultedfrom any one, or a combination,of severalpossible factors (Slosseret al. 1989). Frequencyoflanding by alatesofsome aphidspecies is knownto be influencedby backgroundcolors (Ferereset al. 1999),although white backgroundsare not known to favor landing comparedto darker backgrounds(Liewehr and Cranshaw l99l). It is also possible that the lower cotton canopy temperaturesthat have been reportedto be associatedwith kaolin heatnent in the Lower Rio GrandeValley of Texas Cufufur 2000) were favorableto aphid'performancefor those aphidsthat did settle. Natural enemiesof the cotton aphid could also have been responsiblefor causingthe differencesin aphid populations,but parasitizedaphid mummieswere not counted,and numbersof aphid predatorssuch as chrysopids,hemerobiids, and coccinellids were not significantly different betweentreatnents. Although the hymenopteranfamilies found in the samplescontain parasitoidspecies (Borror et d. 1989),those speciesknown to attack aphidsin particular were not countedseparately. We suggestthat further studieson the mechanismsof cotton aphid population dynamics in relation to kaolin particle film on plant surfacesmight revealopportunities for their management. The lack of changein silverleaf whitefly populations is consistentwith a no-choice laboratory assayusing melon leavesthat showedthat numbersof eggs and adults were not different between kaolin treated and control (water treated) leaves (Liang and Liu 2002). However, the same study showedthat when whiteflies were allowed to choose among leaves treated with kaolin and control leaves, sigrificant differences were observed. It is likely that the plots in our study were of sufficient axea,and the whiteflies did not move sufficiently betweentreated and nontreatedplots, to conform to the no- choiceassay on the melonleaves (Liang and Liu 2002). Populationsof cicadellids were greaterin 2000, and effects of treatmentswere more pronouncedthan during 2001. Nevertheless,cicadellids were more numerous in the kaolin-free contols than in either kaolin treatment during the last month of the two- month samplingperiod in both years. The mechanismfor the suppressionof cicadellid populationsin the kaolin freatnents was not determinedduring this study. Glenn et al. (1999)found that the tarsi ofpear psyllidsin teated orchardswere coated with particles, 143 and this may have induced them to move away from treated areas. The same study determinedthat potato leafhopperfeeding damageon orchard crop leaveswas reduced wherekaolin had beenapplied, but no explanationfor this observationwas advanced. Trendsin the numbersofdipterans were not consistent,althougb in 2000 and in 2001, greaternumbers of dipteranswere found in dvac samplestaken from the connol plots than i1 either kaolin teaunent during the early part of the sampling period. Iater in 2000, populationsin the weekly kaolin treatnent were greaterthan in thi biweekly kaolin featnent. Someofthe changesin dipteranpopulations over time during eachyear could haveresulted from changesin speciescomposition over time, but becauiedipterans were not sortedby species,this is not certain. The interactionin 2001 appearsto be a result of a higher dipteran population in the control plots in late Aprii which declined to abundancesobserved in eachof the two kaolin teatments by early May. The greaternumbers of Orius spp. in the control plot than in either kaolin teatnent was not associatedwith differencesin prey populations,so the kaolin itself appearsto have visually or tactually affeeted their preference. Populationswere greatestin mid- May or early June,a possiblelag after the high cotton aphid populationsin late April and early May. The interactionin 2001 is probably a result of the higher populationfound on 23 Apil in the weekly kaolin heatrrent than in the other two treatments,but on the remaining sampling dates,populations were not sigrificantly different betweenthe three treaunents. Wasp populations differed little throughout the sampling period, and the higher numbersfound on one samplingdate in the biweekly kaolin treatmentdoes not appearto representa frend. Kaolrn was shown to deter boll weevils Aom feeong on excrsedcotton squaresin choice and no-choiceassays, on squ[es on cagedwhole plants, on kaolin treatedfoliage, and on sqrx[es in small plots sprayed with kaolin comparedto non-treated controls (Showler 2002a). The samestudy showedthat when rain did not disrupt the protection provided by weekly kaolin application,cotton lint yields were greaterthan in kaolin-free control plots. Kaolin also deters beet annyworm oviposition on cotton, and it causes 100% mortality to first instars, x60o/omortality to late secondinstars, and protection of treatedleaves against third instars (Showler 2002b). Studieson apple,pear, and cotton foliage indicate that kaolin does not impede plant water potential, light reception,and photosyntheticactivity (Glenn et al. 1999,Showler 2002c). If a way of keepingkaolin on leaf surfaces during rainfall is developed, kaolin could become a less toxic pest managementtool in cotton where use of conventional pesticides is prohibited or undesirable. ACKNOWLEDGMENT Thanksto Jaime Cavazos,Rarll Cant0, Jessede Anda Frank Garcia, Robert Campos, Nancy Rivera, Raul Vallejo, Martin Ponas, Mohamed Osman, Abratra Gana, Andy Cruz, Jaime Lun4 and Martin Galvan for their assistancein the laboratory, field, and greenhouse;and to Tong-Xian Liu and JohnJifon for critical reviews. LITERATTJRECITED Analytical Software. 1998, Statistix for Windows. Analytical Software, Tallahassee, FL. Boyce, A. M. l9?2. Mortality of Rhagioletis completaCress. through the ingestionof certainsolid materials.J. Econ.Entomol. 25: 1053-1059. David, W. A. L., and B. O. C. Gardiner. 1950. Factorsinfluencing the actionof dust insecticides.Bull. Entomol.Res.4l: l-51. 144 Bonor, D. J., C. A. Triplehom,and N. F. Johnson.1989. An introductionto the studyof insects.Harcourt Brace College Publishers, New York, NY, Ebeling, W., and R. E. Wagner. 1959. Rapid desiccationof drywood terrniteswith inert sorptive dustsand other substances.J. Econ. Entomol. 52:.190'207. Fereres,A., G. E. Kampmeier,and M. E. Irwin. 1999. Aphid attractionand preference for soybeanand pepper plants infected with potyviridae. Ann. Entomol. Soc. Amer.92:542-548. Glenn, D. M., G. J. Puterka,T. Vanderzrvet,R. E. Byers, and C' Feldman. 1999. Hydrophobic particle films: a new paradigm for suppressionof artluopod pests andplant diseases.J. Econ. Entomol. 92: 7 59-77l. Harben, P. W. 1995. The industrial minerals handbook II: a guide to markets, specifications,and prices. Arby Industrial Minerals Division Metal Bulletin. PLC, London. Hunt, C. R. 1947. Toxicity of insecticide dust diluents and carriers to larvae of the Mexicanbean beetle. J. Econ.Entomol40:.215-219. Kalmus, H. 1944. Action of inert dustson insects.Science 153 : 7 | 4-71 5. Kennedy,J. S., C. O. Booth, and W. J. S. Kerhaw. 1961. Host finding by aphidsin the field. III. Visual athaction.Ann. Appl. Biol. 49: l-21. Knight, A. L., T. R. Unruh, B. A. Christianson,G. J. Puterka,and D. M. Glenn. 2000. Effects of kaolin-basedparticle films on obliquebandedleafroller, Choristoneura rosacec,nct(Hanis) (Lepidoptera:Tortricidae). J. Econ. Entomol. 93:744-749. Kring, J. B. 1962. Reactionof aphidsto reflectedlight. Bull. Entomol.Soc. Amer. 8: 159. Lapointe, S. L. 2000. Particle film deters oviposition by Diaprepesabbreviatus (Coleoptera:Curculionidae). J. Econ.Entomol. 93 : 1459-1463. Liang, G., and T. X. Liu. 2002. Repellencyof a kaolin particle film, Sunound, and a mineral oil, Sunspray Oil, to silverleaf whitefly (Homoptera: Aleyrodidae) on melonin the laboratory.J. Econ.Entomol. 95:.317-324. Liewehr, D. J, and W. S. Cranshaw. 1991. Alate aphid trap captureover different backgroundcolors and different backgroundpatterns. Southwest.Entomol. 16: 13-18. Makus, D. J. 2000. Cotton performanceas affected by particle film and mycorrhizae treatments,pp. 703-706. 1z Proceedings,Beltwide Cotton Conferences,Ntl. CottonCouncil, Memphis, TN. Puterka,G. J., D. M. Glenn, D. G. Sekutowski,T. R. Unruh, and S. K, Jones. 2000. Progresstoward liquid formulationsof particle films for insectand diseasecontrol in pear. Environ.Entomol. 29:329-339. Richardson,C. H., and L. H. Glover. 1932. Some effects of certain "inert" and toxic substancesupon the l2-spotted cucumberbeetle, D. duodecempunctata(Fab.). J. Econ.Entomol. 25: I 176-ll8l. Showler,A. T. 2002a. Effects of kaolin-basedparticle film application on boll weevil (Coleoptera:Curculionidae) injury to cotton. J. Econ.Entomol 95: 75 4-762. Showler, A. T. 2002b. Effects of kaolin particle film on beet armyworm, Spodoptera exigua (Httbner) (Lepidoptera: Noctuidae), oviposition and larval feeding and developmenton cotton, Gossypiumhirsutum L. Agric., Ecosyst.,Environ. 95: 265-271. Showler, A. T. 2002c. Effeots of water defioit sfress,shade, weed competition, and kaolin particle film on selectedfoliar free amino acids accumulationsin cofton, Gossypiumhirsutum L. J. Chem.Ecol. 28: 615-635. Slosser,J. 8., W. E. Pinchak,and D. R. Rummel. 1989. A review of known and potential factors affecting the population dynamics of the cotton aphid. Southwest,Entomol. 14: 301-313. t45 Unruh, T. R., A. L. Knight, J. Uptorl D. M. Glenn, and G. J. puterka. 2000. particle films for suppressionof the codling moth (cydia pomonella [L.]) in apple and pearorchards. J. Econ.Entomol. 93:737-'143. 146 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 LABORATORY TOXICITY OF INSECTICIDE RESIDUESTO SWEETPOTATO WHITEFLY (HOMOPTERA: ALEYRODIDAE) EGGS,NYMPHS AND ADULTS ON SWEET POTATO, CABBAGE, AND COTTON G. W. EIZEN USDA, ARS, Kika de laGwza SubtropicalAgricultural ResearchCenter, BeneficialInsects Research Unit, 2413 E. Hwy 83, Weslaco,Texas 78596 ABSTRACT Eggs, nymphs, and adults of the sweetpotatowhitefly biotlpe B, Bemisia tabaci (Germadius),were exposed to selectedinsecticides and insect growth regulatorsusing a foliar insecticideresidue bioassay. Thiamethoxam,chlorfenapyr, and imidacloprid were more efficacious materials on B. tabaci eggs on sweet potato than on cabbageor cotton. Imidacloprid was much more effective than other treafinentsagainst eggs on cabbage; tebufenozideand imidaclopridwere moreeffective teatments on cotton than on cabbageor sweetpotato. The insect grofih regulatortebufenozide was more effective againstnymphs thaneggs. Similarly, most of the insecticideswere more effective againstadults on all host plantsthan againsteggs. However, severalteabnents were lesseffective againstadults on cotton versussweet potato and cabbage. INTRODUCTION The sweetpotatowhitefly biotlpe B, Bemisia lcDcci (Gennadius) (Homoptera: Aleyrodidae), has many hosts including cottoq melons, vegetables,and ornameirtals. Henneberryet al. (2000)estimated crop damagecaused by.B. tabaci in California"Arizona, Texas,and Florida to exceed$200 million annuallyfrom the late 1980'sto 1990'sin field and greenhouseproduction. The primary meansfor managingB. tabaci has been the use of insecticides,which has resulted in the developmentof resistanceto all major insecticide classes,including p)'rethroids, organophosphates, carbamates, cyclodienes, and insect growth regulators(Denholm et al. 1996). A new systemicinsecticide, imidacloprid, has provided excellentcontol of B. tabaci on melonand other crops. Howeve4 B, tabaci resistanceto this insecticidehas already been detected in Spain(Catrill et al. I 996). Thereare serious concenul about the developmentof insecticideresistance to other, new classesof insecticidesby 8. tabaci (Prabhakt et al. 1997). Control progr.rmsshould incorporate a diverse array of chemicalinsecticides from a variety of chemicalclasses. The objective of this studywasto determinethe toxicity ofconventional,biorational, and newerinsecticides for controlof immatureand adult stagesof .8. tabaci onthreedifferent host plant species. MATERIALS AND METHODS A laboratoryspray chamber @eVries MFG, Hollandale,MN) was usedfor all spray applicationsfollowing the methodsof Elzenet al. (1998). Air was exhaustedfrom the spray chamberthrough a built-in exhaustfan andthe chanrberand spraysystem were washedwith 147 water betweentreatments. Pressure was 1.81kglcm2 , using threehollow conenozzles (TeeJetTXVS-6, TeeJetSpraying systerns, wheaton, IL), anda boom speedof 4.g km/h. This systemapplied 280 liters/ha. The nozzleswere anangedto optimize coverageto the undersidesof the plants using a cenhal nozzlewith two more nozzleson dropsto the sides. The dropnozzles ran nearthe level ofthe chanrberfloor but weredirected upward toward the undersideofthe leaves.Formulated insecticides, diluted to recommendedfieldrates (Norman andSparks 1997, Sparks 2000), or from manufacturer'srecommendations in the caseofnon- registeredmaterial, were used as treatments. Water was used as a control treatrnent. Formulatedinsecticides tested were endosulfan [Phaser 3 suspensionconcmtate (sc); Bay€r, Researchrriangle Park,NCl, abamectin[Agri-mek 0.15 emulsifiableconcentrate (EC); Syngenta,Greensboro, NCl, thiamethoxam [CGA 293343 24 wettable granule (WG); Syngenta,Greensboro, NC], tebufenozide[Confirm 2 flowable (F); Dow AgroSciences, Indianapolis,IN], imidacloprid[Provado 1.6 F; Bayer,Kansas City, MO], chlorfenapyr [Pirate3 solubleconcentrate (SC); Bayer,Research Triangle Park, NC], pymetrozine[Fulfill 50 WG; Syngent4 Greensboro,NCl, and buprofezin[Applaud 70 wettablepowder (WP); Bayer,Research Triangle Park, NC]. Ratesused are shownin Tables l-3. Plantswere grownin 22-cmplastic pots filled with Sunshine#3 soil mix (SunGro HorticultureInc., Bellvue,WA). Sweetpotato (Ipomoea batatas L.), cabbage(Brassica 'Cheers'), oleraceaL., or cotton (Gossypiumhirsutum L., 'Sure-Grow125) to be testedwere placedin a greenhousewith a high populationof.B. tabaci on greenhousetomatoes for 48 h. Plants were taken to the laboratory and one leaf per plant with tbree replicatesper plant specieswere selected.Forty-five B. tabaci eggsper leafwere selectedand treatedwith the insecticides.Eggs were enclosedin 3-cm diameterclip cagesand held for l0 dayswhen mortality was determined.For testingnymphs, plants were similarly infestedwith eggs. Ten to twelve days after infestation, eggshatched and 45 nymphs per leafwere selectedand freatedwith insecticides.Clip cageswere appliedto eachleaf and mortality was determined 72h afterf.eatment. For adult tests,infested plants were treatedwith the spraychamber as above.One leafper plant with four replicatesper plant specieswas selected,and 25-30 adults were countedand placedunder a clip cage. Mortality was determinedT2h after treatrnent. Control mortality was never greaterthan 10.0%;data were conectedfor conEol mortality usingAbbon's (1925)formula- Percentagemortalities were acrsine fansformed and arralyzed by analysisofvariance; means were separatedusing least significant difference [P < 0.05 (SASInstitute 1988)1. RESTILTSAND DISCUSSION Thiamethoxam,chlorfenapyr, and imidaclopridwere the more efficaciousmaterials on B. tabaci eggson sweetpotato. Imidaclopridwas much more effectivethan other ffeabnentsagainst eggs on cabbage;tebufenozide and imidacloprid were the more effective treatmentson cotton (Table l). The insectgrowth regulatortebufenozide was sigrificantly moreeffective against nymphs than eggs,as might be expected(Table 2). Similarly, most of the insecticideswere more effective against adults on all host plants than againsteggs. However,several heatnents were lesseffective against adults on cotton versussweet potato andcabbage (Table 3). In general,imidacloprid and thiamethoxanwere more effective on any life stageon sweetpotato than othertreafin€lrts. Imidacloprid and thiamethoxarnw€re more effective on cotton; however, otler chemials were very effective, particularly on nymphs and adults. Imidaclopridwas also an effective treatmenton cabbage,but other treatmentswere equally effective on nymphsand adults. r48 TABLEI. ToxicityofSelectedlnsecticidestoB.tabaciBggsinaFoliarlnsecticideResidue Bioassayon Three Plant Species,72 h Post-feafinenl o/^ Mortnlitv dts2,6 F= Treatme,nt Ko (ATl/ha) Sweet Poteto Cabbase Cotton Abamectin 0.01I 20.7abA 2l.2aA l2.6a[ 0.57 Tebufenozide 0.28 1l.9aA 34.1aAB 60.7b8 4.3r Imidaclo,prid 0.052 33.9bA 92.6b8 6l.5bB 11.13 Chlorfenryyr 0.39 36.3bA 18.5aA 49.6ab[ 2.93 Thianetlroxam 0.051 45.9bA' 20.OaA 28.2ab[ 2.76 Pymehozine 0.096 2.9a4 l9.3aB 34.lab0 28.78 Endosulfan l.t0 l4.laA 22.2a4 39.2abA 2.80 Bupmfezin 0.39 2.9aA. l9.3aA 2l.5aA. 1.93 F=3.55 F=24.2 F=I.99 dH7,16 dts,7,t6 dF'!t,16 Yalues within a column followed by the samelowercase letter or within a row followed by the sameuppercase letter arenot sigrificanfly ditr€reNil(P > 0.05; leastsignificant difference [SASInstitute 19980. TABLE 2. Toxicity of SelectedInsecticides to B. tabdci Nymphs in a Foliar Insecticide ResidueBioassay on Three Plant Species,72 h Post-teatnent. o/" MortaliM dF2,6 F= Treaffirent Kg (AT/ha) SweetPotato Cahhage Cotton Abanectin 0.011 65.9abA 58.5abA 28.9aA 3.21 Tebufenozide 0.28 38.5aA 6l.5abB 79.3abB 6.95 Imidacloprid 0.052 92.6c4 98.5cA 88.9bA 0.65 Chlorfenpyr 0.39 84.5bcA 89.6bcA 88.9bA 0.07 Thisnettroxam 0.051 80.0bcA 63.7abcA 73.3ab[ 0.10 Pymetozine 0.096 59.3abAB 46.7a4 87.4bB 3.50 Endosulfan 1.10 83.0bcA 93.3bcA 83.7bA 0.43 Buprofezin 0.39 763b4 75.6abcA 88.9bA 0.59 F4.69 F-2.36 F=I.65 dF7,r6 dts,7,16 dF{7,16 "Valueswithin a column followed by the samelowercase letter or within a row followed by the sameuppercase letter arenot significantlydifferent (P > 0.05; leastsignificant difference [SAS Lrstitute1998]). t49 Imidaclopridand thiamethoxambelong to the samegeneral class of insecticides. Thiamethoxamis a second-generationneonicotinoid compormd with stomachand contact activity, like imidacloprid. Thiamethoxambelongs to the subclassthianicotinyl of the neonicotinoid insecticides,which, like imidacloprid, interferes with the nicotinic acetylcholinereceptors in the insect nervoussystem (Maiensfisch et al. I 999); therefore,it is not surprisingthat thesecompounds would havesimilar activity. Thesechemicals are more selectivelytoxic to insectsthan warm-bloodedanimals. Thiamethoxamhas been found to be low to moderatein toxicity to at leastone parasitoid, Aphelinis gossypii Timberlake, but high in toxicity to the whitefly predator Delphastuspusillus (LeConte) (Tones et al. 2003). Imidacloprid has been widely used for contol of B, tabaci, rusrng concernsabout developmentofresistance to this insecticide(Stansly et al. 1998,Cahill et al. 1996). TABLE 3. Toxicity of SelectedInsecticides to B. taDaciAults in a Foliar InsecticideResidue Bioassayon Three Plant Species,72 h Post-treatrrent. o/^Il vy''ortalitv df-.'2,9 F: Treafmeni Ko lAT\/hn\ Swee.f Pnfaln Cahhaoe Abamectin 0.011 86.7b8 87.5aB 40.8aA 5.69 Tebufenozide 0.28 50.0abA 76.7a4 33.3aA 2.07 Imidacloprid 0.0s2 M.2ab[ 83.3aA 52.5a4 1.83 Chlorfenapyr 0.39 86.7b8 87.5aAB 52.5a4 2.99 Thiamethoxam 0.051 86.7bB 87.5a8 46.7aA 4.18 Pymetrozine 0.096 23.3a4 85.8a8 28.3a4 5.78 Endosulfan l.t0 86,7b8 87.5aAB 52.5a4 2.99 Buprofezin 0.39 68.3abA 86.7aA 325aA 2.07 F+..27 F4.77 F=-0.23 df=4,U dF7,U dF,7,24 Yalues within a column followed by the samelowercase letter or within a row followed by the sameuppercase letter arenot sigrificantly different(P > 0.05; leastsigrrificant difference [SASInstitute 1998]). The finding that pymetrozinewas not very toxic to eggsis not surprising. It has a novel modeof actionwith selectiveactivity againsthomopteran insects like the cotton aphid and the whitefly in cotton (Nicholsonet al. 1995). Pymehozineinterferes with neryous regulation of feedingbehvior, which consequentlyresults in deathdue to starvationafter a few days(Nicholson et al. 1995). Pymetrozinewas found to be harmlessto A. gossypiimd D. pusillus (Tones et al, 2003) and may havea fit for controlling 8. tabaciwhile providing new chemistryto delaythe developmentofresistance to neonicotoidinsecticides. Insectgrowth regulators, such as buprofezin and tebufenozide, which selectivelytarget B. argentifolii and not their natural enemies,have been successfullyused for controlling nrunerouspest insects. They have been used against B. tabaci on cottanand melons (Dennehy andWilliams 1997)and showed good eflicacy in thepresent shrdy, Hostplants (i.e. plant allelochemicals)can modiff the susceptibilityof herbivorous arthropodsto pesticides(Yu 1986,Brattsten 1988). However,in the caseherein, B. tabaci werealways reared on tomatoand were onlyplaced on the threeplant speciesfor a shorttime. This doesnot rule out the possiblity that therewere chemicalor physicalinteractions which 150 would be a factor in the results. ln this case,the mobile stagesof B. tabaci wete generally more zusce,ptibleto the insecticidestested' Furtherstudy may be warranted In recentyears, efforts to improve chernicalconhol of .8. tabacihave focusedon the evaluationof slntemicinsecticides, insect growth regulatorc, and biopesticides. A significant arnountof work was repq4edon the developmentof usepattems for the neonicitinoid class of chemistry. Thiamethoxam has provided an altemative to the prophylactic use of imidacloprid. Use of insect growth regulators remains an important chemical control approach@razzle 2OO0). It appearsfrom the data that chemicaltreatments for B. tabaci should be timed to target nymphs and adults. Also, insect growth regulators and biorational pesticides,in agreementwith the above rationale, could provide alternativesto conventionalchemical pesticidesfor effective confrol, while preservinginsect natural enemies. LITERATURB CITED Abbott, W. S. 1925. A methodof computingthe effectivenessof an insecticide.J. Econ. Entomol. 18:265-267. Brattsten,L. B. 1988. Potentialrole of plant allelochernicalsin the developmentof insecticideresistance, pp. 313-348.In P. Barbosaand D. Letoumeau[eds.], Novel aspectsof insectplant interactions. Wiley, New York. Brau,le,J. 2000. Chemicalcontrol, biopesticides,resistance managernent, and application methods,pp.72. In T, J. Hermeberry,R. M. Faust,W. A. Jones,and T. M. Peming [eds], Silverleafwhietfly: National research,action, and technolory tansfer plan (formerly sweetpotatowhitefly, strainB); Third annualreview of the second5-year plan. U, S. Departrnentof Agriculture, Beltsville, MD. Cahill, M., K. Gorman,S. Day, andI. Denholm. 1996. Baselinedetermination and detection of resistanceto imidacloprid,in Bemisia tabaci (Homoptera:Aleyrodidae). Bull. Entomol.Res. 86: 343-349. Denholm,I., M. Cahill,F. J. Byme,and A. L. Devonshire.1996. Progress with documenting andcombating insecticide resistance in Bemisia,pp.577-603. /n D. Gerlingand R. T. Mayer [eds.'jBemisia 1995:taxonomy, biolory, damage,control andmanagement. htercept Ltd., Andover, Hants,UK. Dennehy,T. J., and L. Williams, m. 1997, Managementof resistancein Benisia in Arizona cotton.Pestic. Sci. 5l:398-406 Elzen,G. W., P. J. Elzen,and E. G. King. 1998. Laboratorytoxicity of insecticideresidues to Orius insidiosus,Geocoris punctipes, Hippodamia convergens, md Chrysoperla carnea.Southwest. Entomol. 23: 335-342. Henneberry,T., R. Faust,W. A. Jones,and T. Perring.[eds.]. 2000. Silverleafwhitefly: NationalRes., Action, andTechnologyTransfer Plan, 1997-2001.July 2000,USDA- ARS. Maiensfich,P., L. Gsell, and A. Rindlisbacher. 1999. Synthesisand insecticidd activity of CGA293 343: a novelbroad-spectrum insecticide. Pestic. Sci. 55: 351-355. Nicholson,W. F., R. Serm,C. R. Flueckiger,and D. Ruog. 1995. Pynehozine:a novel compoundfor conholof whiteflies,pp, 635-639..ln D. Gerlingand D. Mayer[eds.]. Bemisia: taxonomy, biology, damage,conhol, and management. InterceptPress, Andover. Norman,J. W., Jr., andA. N. Sparks,Jr. 1997. Suggestedinsecticides for managementof cottoninsects in thelower Rio GrandeValley. Tx, Agric. Ext. Serv.Bull. B-l2l0A. Prabhaker,N. N., N. Toscano,S. Castle,and T. Henneberry. 1997. Selectionfor imidacloprid resistance in silverleaf whiteflies from the Imperial Valley and 151 developmentof a hydroponicbioassay for resistancemonitoring. Pestic.Sci. 5 l : 419- 428. sAS Institute. 1988. sAS/srAT user's guide, version 6.03 et. sAS Institute, cry, NC. Sparks,A. N., Jr. 2000. Tsras guide forco'ntrolling insectsof commercialvegetablecrops. Tx. Agric. Ext. Serv.Bull. B-1305. stansly,P. A., T.-x. Liu, and c. S. vawina. 1998. Responseof Bemuia argentifotii Homoptera: Aleyrodidae) to imidacloprid uder gfe€nhouse,field, and laboratory conditions. J. Econ. Entomol. 9l:686-692. Torres,J. B., c. s. A. Silva-Torres,and J. vargasde oliveira. 2003. Toxicityofpynetozine and thiamethoxamto Aphelinusgossypii andDelphastus pusillus. pesq. agropec. bras.,Brasilia. 38: 459-466. Yu' S. J' 1986. consequencesof induced foreign compound-metabolizingenzymes in insects,pp. 153-174.InL.B. Brattstenand S. Ahmad[eds.], Molecular aspects of insect-plantassociations. Plenum, New York. 152 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 EVALUATION OF FEEDING STIMI.JLA}ITS COMBINED WITH POLYMERS TO DEVELOP FORMLILATIONS AGAINST DUTMEA SACCHAMLIS F)l Ninfr M. Rosas-Garcfa,Katiudrka Ar6valo-Niffo,Luis J. Galdn-Wottg md Lilia H. Morales-Ramos2 Departamentode Microbiologla e Inmunologia,Facuftad de CienciasBiol6gicas, U.A.N.L., A.P.2790. SanNicol6sde los Garz4N. L. M6xico.C.P. 66450. [email protected]<. ABSTRACT Three fteding stimuhnts (sucrose, powdered sugarcang md Coo RESUMEN Dirrcrsascombinaciones de tres fuoestimulantes (mcarosa cafia de aalcar en polvo y Coar@ y tres pollnrros biodegradables(gelatina, pectina y almid6nde rralfzrnodificado) fueron elaboradaspara desarrollar fomnrlacionesgranulares y obtener la mfs adecuada para las larvas de Diatraea saccharqlis (F). Las larvas ingirieron preferentementelos grdnulos que codenfm gelatina o pectina. t os frgoestimulantescausaron diferentes efectosen la palatabilidad siendoel m6s firerte b catu de adrcat en polvo, seguidopor la srrarosa y finalrnerrtepor el Coax@.[p5 nn4lisisestadisticos indicaron que la formulaci6n nrds aceptadapor D. saccharalisfue la mezclade almid6nde miz rnodifioado,gelatina y cafiade azfcar en polvo. INTRODUCTION The sugarcaneborer, Diatraea saccharulis(F.), is one of the most importantpests of the sugarcaneproducing regions of Mexico. The damageis causedinitially by neonate larvae whbh feed on the surfrce of foliage and then tunnel vertically within stalks,uAere they remain through pupation, weakening the plant and causing with€ring and death (Davidson199). I Lepidopthera :Pyralidae. ' To whom reprint request slrould addressed. 153 The sugarrcaneborer damagesup to 3v/o of sugarcaneintemodes; while in their galerigs, lanfe ar€ protected from climate and insecticides.over the last 40 years,much researchhas beenconducted to frrd viable altemativesto control this pest but resultshave been negative(Grupo Azucarero Mdxico 198). The use of chemicafinsecticides has not been satisfictory because insectici MATERIALS AND METHODS Pupae of D. soccharulis were provided by USDA Weslaco, Tq r54 TABLE l. Combincion ofBiodegradablePolyrers and FeedingStimilants. Formulatbn Pobrnrrs I€eotng $murams Modified Cr€latin P€ctin ffi corostarch sugaroane F2 x F3 x T F4 x F5 x x T F6 x x w x x x F8 x x F9 x -x i F10 x -x x Fll x -x x Ft2 x -x FIG. l. Two ohoil:e bioassays:A) Conpcison betwe€n two formrldions, and B) Coryarison betweena shgle fornulation ad a pitrceof freshsugarcao€. Each formrlatbn was rehydratedin p€tri dfubpsrmtil tbcy becam soft and rubbery. Ten 2-day old larvae wpre placedb tbe ceder of eac,hdislu whic,hwas ceped and seal€d tighly with Paafitnrx. P€tri disb€swere then placed in thc da* at 28oC for 16h, after' nfiich the dishesw€re placedin afrer.zs at -l6oC for 8h to terminatethe test accordingto Bartelt et al. 1990. Obsenrationsindicated that larvac did not leave their feeding sites as tbe dish wenecooling. The nrnbers of lanne d the two f€ediry sites were rpcorded. Corryarisom were ma& in pairs with fi/e r€plicat€s.Tbe rcsults ohained in the ftding preferencebbassays we,r,e nrb,mitted to AI{OVA and Tukey Test for ntem coqarisons withP S 0.05. 155 RESI.]LTSA}ID DISCUSSION We obtained 12 different formulations, all of which included modified cornstarch becauseit is considereda protectirrc ag€nt ofthe toxic activity. Also, gelatin and pectin were used for the saurc purpos€ becausethey are palatable to larvae, as reported previously by Morales et al. (1998). The formulationswere grouped accordingto tbeir Ming stfunulantcontent (Table 2). The formulatbm containing feeding stfurrlflfs and those without feeding stimulails were coqared; resuhs sbow a higbly significd ditrerencebetween Group I ard Groups2,3, ad 4, (F = 24.65,df = l, P S 0.01) (table 3). Tbesedata show that the addition ofa feedingstimulant increases preference by lallae. Feedingstimulants were also coryared, and they showeda highly significant differerrce. Powdered sugarcanewas the most preferredfteding stimulant, followed by zucroseand then Coaxil (Table 3). TABLE 2. GroupsofFormulations Accordingto FeedingStimulant Content. Group Content Fornnrlmions I No FeedingStimutad FI F5 F9 2 Coax F2 F6 Fl0 3 Sucrose F3 m Fll 4 PowderedSugarcane F4 F8 Ft2 TABLE 3. Anal;ilsisof Variance Obtainedfrom the Coryarison of the Forrrulationswith Ditrer€NrtFeeding Stimulants. Coryarisons MeanNo. larrae on groups ANOVA betweengroups +SE GroupI vsGroups 2, 3,4. 2.22+0.10vs 1.3 + 0.10 F =24.65,df= 1,P < 0.01 Group 2 vs Group 4 1.47+0.12vs3,96+0.19 F= 113.48,df=l,P< 0.01 Group 4 vs Group 3 2.71+0.13vs 1.22 + 0.10 F:54.16,df= I, PS 0.01 Group 2 vs Group 3 1.47+0.12vs2.59 +0.13 F =29.14,df=I, P< 0.01 Freshsugarcane pieces" 8'65+0'l8vs1'99+0'08 F=534'93'df: l,P< 0'01 Allgroups Mean corparisons indicatedthrt F8 and Fl2 formulationswere the most preferred by larvae (TaUe a). Freshsugarcane served as a positive control and was more preferred th4n any formulation. Basedon theserezults, we selectedth€ F8 bl€od as the most suitable formnlatbn for D. sacchualis larvre. Our resultssbowed that the pres€nceof feedingstimlms incr€asedpreference, od demonstratedthat larva€ preferred the blends basedon modfied cornstarcb gelatin or pectin ard powderedsugarcane. Ahhough there was no sipificant differencebetween F8 aadFl2 formulations,the F8 fomulation was selectedas the most suitablesince gelatin is inexpensive,widely arrailable,and easyto use. 156 We obeerved rbart larvae rarely ftd on grarular formrhtions containing only rnodified comstarclg larnae preferred those with geldin and pectin combined with rnodifed comstarch. We rnaintain that modified comstarch ir an iryortad ingredient becauseit protectsinsecticidal activity arxl givesproducts an acceptableshelflife. TABLE 4. Mean Conparison Test for All Formulatiens. Meanrumber m Formtrlatioru eachformulation sE' FI 0.86+ 0.1 " F2 0.58+ 0.11 F3 0.81+ 0.14"b F4 2.gt *o.2gd F5 1.23+ 0.16'bc F6 1.88+ 0.25bd F7 1.50+ 0.17sb" F8 4.80+ 0.36u F9 l.g0 + 0.23h' Ft0 l.gS+0.23c4 Flr 1.35+ 0.20lt" Ft2 4.18r 031 € Piecesoffresh nt' TirkeY b= Test, P S 0.05. Pi:ces offiesh sugrcane were usedas a control Powderedsugarcan€ was the rmst preferredftding stimulail. Susros€was a mild feeding stimrlam in ou study. Our r€sufts agree wiih those of Barteh et d. (1990) ad Gillispie et al. (1994) who reported that g;lucoseand rnolassespossessed ht€rmediate palatabilityto Osbinia wbilalis (HUbner).Tbe cormtercial feeding stimulantCoo ACKNO1VLEDGMENT The authorse:press theh gratitudeto Vfotor Muuel Rosas-GarcfaSergio Salc.edo- Martfnea Arcadio Valdds and JosdLuis Ochoa for tleir excellentcritical reviews of this mmusoript. They also thank the United Stales Oeearmm of Agriculture, Weslaoo, Toras, for providing pupae of D. sacclwralis: the United States Deputnent of Agriculture, Peoria lllinois, for providing CootrM; and Aranal, S.A de C.V, Monterre' , N.L., Mexico, for providing modifed comstarch This study received fnancial support fiom tbe Consejo Nacional de Ckrcia y Tecnobgia (CONACyT) Project No.29365B. Ninfr M. RosarGarcia is a recipient of a ftllowship fiom the Consejo Nacional de Cienciay Tecnologfa(CONACyT). r57 LITERATURE CITED Bartelt, R. J., M. R. McGuire, and D. A. Black. 1990.Feeding stimulants for the Eruopean com borer (Irpidoptera:Pyralidae): Additives to a starch-basedformulation for Bocillus tlnringiezsis. Em,hon Entomol. 19: 182-189. Davison, R. H. 9m. plagac de pastos y cerealsnpp 189-207.12 -plagasde Insectos furicolas y del JardirnNoriega [ed.]. Limusa.MCxico. Dunkle, R L., and B. s. shasha 1988. starch-encap$rhtd Baciilus thuringiensis:A potential new method for increasingenvironrnental stability ofentomopathogens. Environ Entornol. 17: 120-126. Farrar, R R, and R L. Ridgway. 1994. co4arative studiesof the effects of nutrient- basedphagostimulants on six lepidopterousinsect pests. J. Econ Entomor.8T: 44- 52. Gillespie, R. L., M. R. McGuire, and B. s. shasha. 1994. palatability of flour granular foruulations to European com borer larvae (Lepidoptera:Pyralidae).l. gcon Entomol.87:452457. Grupo Azucarero M6dco. 1998. Anteproyecto de programapara el control del gusano barrenador del tallo en el estado de Sinaloa. Docunrento de trabajo. Gerencia corporativade campo. McGuire, M. R, B. S. Shash4 L. C. I€wis, R J. Bartelt, urd K Kinrrcy. 1990. Field evaluation of granular starch formulations of Bacillus thuringiensis against Ostrinianubilalis (Lepidoptera:$ralidae).J. Econ. Entomol. 83:2207-2210. McGuire, M. R., qnd B. S. Shasha 1992. Adlg€nt starch granulesfor encapsulationof insectcontrol agents. J. Econ Entornol.85: 1425-1433. McGuire. M. R., B. S. Sbastt4L. C. Lewis, and T. C. Nelsen. 1994. Residualactivity of granular starch-encapsulatedBacillus tlnningiensis. J. Econ. Entomol 872 631- 637. McGuire, M. R., B. S. Shash4C. E. Eastnralr,and H. Oloumi-Sadeghi.1996. Starch-and flour-based sprayableformulations: Effect on rainfastnessand solar stabiliry of Bacillw thuringiensis.J. Econ Entornol.89: 863-869. Morales Ramos,L. H., M. R McGuire, and L. J. Gal6nWong. 1998.Utilization of serrcral biopolyners of granular formulationsof Bacillus thuringiensis.J. Econ. Entomol. 9l:1109-1113. Morales-Rarnos,L. H., M. R McGuire, L. J. Cral6n-Wong,and R. Casno-Franco.2000. Evaluation of pectin, gelatin and starch granular forrrulations of Bacillus SoutlwesternEntomologist. 25 : 59-67. Ridgway, R. L., V. L. Illuru R. R Farrr, JR., D. D. Calvi& S. J. Fleischer,and M. N. Inscoe. 19!)6.Granular matrix forrnrlation of Bocillus tlruringiensisfor cotrol of the Europeancorn borer (Lepidoptera:Pyralidae).J. Econ Entomol. 89: 1088-1094. Rosas-Garci4 N. M. 2002. Elaboraci6n de formulados de Bacilfus thuringiensis ver'. hrstaki y determinaci6o de la activftlad t6xica contra hrvas de Diatraea saccluralis (Fabricius) (kpidoptera:Pyralidae) en laboratorio y campo. Tesis de doctorado.F.C.B., U.A.N.L. Monterrey,N.L. Mdxico. ShoreS H. H. 1963. A sirrple artificial rearing medium for the cabbagelooper. J. Econ Erfomol 56:53G537. Wiedennanq R. N., and J. W. Smith, Jr. 1993.Fwrtional resporse ofthe parasiteCotesia flotipes (Hymenoptera: Braconidae) at low densities of the host Diatraea saccharalis(Lepidoptera: Pyralidae). Environ EntorcL 22: M9-858. WiedenmannR N., and J. W. Snrith, Jr. 1995. Parasitizationof Diatraea sacchoalis (Lspidopt€rd:Bmlidae) by Cotesia chilonis and C. flwipes (Hyrnenoptera:Braconidae).Environ Entomol. 24:.95G%1. 158 vol.29 NO.2 SOUTHWESTERNENTOMOLOGIST JUN.2004 MINUTES OF THE 2OO4ANNUAL MEETING OF THE EXECUTIVECOMMITTEE OF THE SOCIETYOF SOUTHWESTERNENTOMOLOGISTS. TheExecutive committeemet at 3:30p.m. on February23,2004,attheHolidayknPark Plaza in Lubbock, Texas, during the Annual Meeting of the SouthwesternBranch of the Entomological Society of America. Presentwere PresidentDrees, Present-Elect Jonathan Edelson,Past-President John Jackman and Secretary-Treasurer Allen Knutson. The Editor's andSecretary-Treasurer's reports were reviewed and approved. Progress to compileall back issuesof theSouthwestem Entomologist onto a CD werediscussed. The Committee approved of the contractwith Bill Samesfor scanningback issuesof the joumal and supplementand authorizedpayment of $500to Bill Samesfor expenses.The ballots for President-Electwere providedto theNominations Committee for counting.The meeting was adjoumed at 4:00 p.m. MINUTES OF THE 2OO4ANNUAL MEETING OF THE SOCIETY OF SOUTHWESTERNENTOMOLOGISTS. TheAnnual Meeting of theSociety was called to orderby PresidentBart Drees at 4:00 p.m. on February23, 2Q04,at the Holiday Inn Park Plazain Lubbock,Texas, during the Annual Meetingof the SouthwesternBranch of theEntomological Society of America.Twenty-two membersof the Societywere present. Tbe minutesof the 2003annual meeting as published in the Juneissue of the SouthwesternEntomologist were distributed and approvedas printed. The Secretary-Treasurer'sreport and Editor's reportwere disffibuted, reviewed andapproved. PresidentDrees reported on progressin makingall ofthe pastissues ofthe Southwestem Entomologistavailable on CD. Bill Sameshas scanned all of the first 25 volumes,and bids arenow out for addinga searchengine to the CD. The costofthe CD was discussedand a motion madeand passed to chargemembers cost plus postageper CD while non-members would be chargedan additional$25.00 per CD. PresidentDrees reported that the changeofthe Society'sname had beenolficially recordedby the Secretaryof Stateand that the filling feeof $25.00had been paid. President Dress further reportedthat the new membershipbrochure had beenprinted and a copy distributedto eachmember along with the membershiprenewal statements. He reportedthat the Society's web page (http://sswe.tamu.edu)was available and that Anna Kjolen, Deparfinentof Entomology,Texas A&M University,had contributedsignificantly to this effort andwould continueto maintainthe site for the Society. PresidentDrees asked members to submit items (e.g., photosand historical documents)for inclusion on the site. Zopheras nodul osushaldemani, the ironcladbeetle, shown on the cover of thejoumal, is now the official commonname for this insect.President Drees reported on work with TopsPrinting to updatethe cover desigr and format of thejoumal. While thedimensions ofthejournal would not change,changes in font, layout,and cover desigr, includingthe optionfor a photographon the cover,were discussed. There would be someinitial costfor developingthe desigt but little additionalprinting cost except possibly for theuse of glossypaper.A motionwas made and passedrecommending the new cover designbe accepted.There was generalagreement that updatingthe j ournal's format would bebeneficial and President Drees agreed to work with Editor DarrellBay to furtherconsider these changes. 159 A supplementdevoted to insecticideefficacytrials was discussed. Those in favornoted that manymembers of the Societyare applied entomologist and such a supplementwould provide an outlet for publishing this work in a refenedjoumal. Othersnoted that Arthropod ManagementTests can meetthat needand that the policyof the SouthwestemEntomologist hasbeen not to publish efficacy trials. It alsowas noted that it would be necessaryto havJan editor for this supplement,provide for the editor's stipend and a policy to set standardsfor acceptingpapers (e.g., recent trials versushials conductedfive or more yearsago). The cunentguideline is thatifthe subjectinsect occurs in thesouthwestem US, then the paper can be consideredfor thejoumal, eventhough the researchmayhave been done in an areaoutside of the southwestemUS. Another suggestionwas to make this supplementan electronic publicationonly availablethough the Society's website. A proposalto publisha supplement on insecticideefficacy Eials was tabled for furtherdiscussion. It was noted that the Society would continue to pay one-half of the awardsfor the StudentPaper Competition in conjunctionwith the SouthwestemBranch of ESA andinclude a one-yearmembership to the Societyfor eachwinning student. The nominatingcommittee, consisting of JohnJackrnan and phillip Mulder, re,ported that a count ofthe ballots found a majority vote for Tom Royer as the new President-Elect. PresidentDress then passedthe gavel to in-comingPresident Jonathan Edelson. Allen Knutsonthen presentedPast-President Dress with a plaquein recognitionfor his servicesto the Society. PresidentEdelsen noted the continueddecline in membershipand that this wasto be expectedbut that thejournal continuesto servea vital needamong entomologists in the southwestenrUnited Statesand Mexico. Therebeing no furtherbusiness, a motionwas made, seconded,and passed to adjourn. RespectfuI ly submitted, Allen Knutson,Secretary-Treasurer 160 SECRETARY-TREASTIRER'SREPORT Februaryl, 2003-JanuarY31, 2004 Balanceon HandFebruary 1,2003 s4,163.73 Income: Memberships $ 3,890.00 SubscriPtions 2,440.00 PageCharges 15,s45.00 RoYalties 148.28 Total Income $22.023.28 Expenses: Joumal: Editor's Fee $2,000.00 Printing 12,604.18 SecretaryFee 2,000.00 Ad Mail Mailing Service 929.58 Postage 1003.26 Supplies 150.00 Society: SecretaryFee $1,000.00 Supplies(Copying) 160.78 Postage 200.10 PresidentPlaque 74.20 Secretary-TreasurerFee 1,500.00 CD ofBack Issue s00.00 MembershipRecruitment Brochure 777.00 Total Expenses s22,5Q9.40 BalanceofHand January31,2004 $3,6' t7.6r As of January3l,2004,there were 323 membersand 98 institutional subscribers to the Societyof SouthwestemEntomologists. Respectfully submitted, Allen Knutson, SecretaryTreasurer l6l EDITOR'SREPORT Therewere a total of 42 manuscripts,comprising 301 pages,published in the four regular issuesof volume 28 of the southwestementomologist-du;ng)003 compareo to 37 manlsgripts and 314 pages in volume 27 for 2002. Additionallyl SupplementNo. 26, consistingof37 pages,and Supplement No. 27,consisting of I +z pages,alslwere published during the year. A total of 63 manuscriptswere received for considerationfor publicationdwing 2003, comparedto 69 during2002. A numberof theseare still in thereview process; however, three have beenrejected as of this time, The total of tlree manuscriptsrejected represents a decreaseoffour from the sevenmanuscripts rejected during 2003, and constitutes a yearly rejectionrate of approximately5%. Editor'sFinancial Report Date Description ReceiptslExpenditures Balance 0l/01/03 BalanceForward $ 47.57 o2/0r/03 Postage 4.93 42.64 02/r5/03 Postage 4.93 37.71 03/03/03 Postage 22.t2 15.59 03/10/03 From Treasurer 100.00 115.59 04/04t03 Postage 9.17 106.42 05124/03 Postage 25.tI 81.31 06/28/03 Postage 18.21 63.10 07/08/03 Postage 19.00 44.10 07/26/03 Postage 2t.60 22.50 08/16/03 Postage 18.52 3.98 09/rs/03 From Treasurer 150.00 153.98 09/06/03 Postage 14.03 139,95 r0/25/03 Postage 23.08 116.87 CashSummary BalanceForward 0l/01/03 s 47.s7 Receipts 250.00 Expenditures 180.70 $116.87 EndingBalance Respectfullysubmitted Danell E. Bay,Editor 162 AUDIT COMMITTEE REPORT I haveexamined the financial records of the Societyof SouthwesternEntomologists andthe Secretary-Treasurer's Report for January,3l2003, through January 30,2004, andthe recordswere found to be in properorder. Respectfully submitted, JonathanEdelson, President Societyof SouthwestemEntomologists 163 vol.29 NO.3 SOUTHWESTERNENTOMOLOGIST SEPT.2OO4 USE OF THE PHEROCON@CRWTRAP TO MONITOR ABTINDANCEOF WESTERN CORNROOTWORM (COLEOPTERA: CHRYSOMELIDAE): ECONOMIC TTIRESHOLDS,TRAP PLACEMENT, AND COMPARISON TO ANOTHER LURE TRAP J. Singh,G. Wilde, R. Shufran,J. Whitworth Departrnentof Entomology,Kansas State University Manhattan,KS 66506-4004 ABSTRACT Studieswere conductedduring a three-yearperiod to determinethe relationship betweennumbers of westemcom rootwormbeetles, Diabrotica virgifera virgiferaLeconte, caughtin a new Pherocon@lure trap and damageby com rootwo-rnlarvaJine following year.Data suggested that an averageof lessthan 200 beetlesper trapper weekresulted in a populationnot causingeconomic loss to roots by larvae the following year. Data also suggestedthat traps on the exterior (outsiderow) ofa com field usually reflectedabundance in the field and could therefore be used in a predictive manner when abundanceof com rootwormsis nearor exceedsthe economicthreshold, Comparison with the Csalomon@lure trap showedthat the Pherocon@CRW trap was more efficiint (traps more beetles)and was easierto handleand change. INTRODUCTION The westerncorn rootworm, Diabrotica virgifera virgifera Leconte,is a major pest of cotn,Zea maysL., in the United States.Reliable and practical monitoring techniquesare neededto ensurethat insecticideto control this pest is needed.Monitoring techniques shouldbe reliableindicators ofpest abundanceand should be acceptableto endusers. Many kinds of monitoringtechniques for westerncorn rootwom adultshave been testedand described during the last few decades(Musick andFairchild 1970,Chiang1973, Luckmannet al. 1975,Hein and rollefson 1984,Shaw et al. 1984,Hoffrnan et al. 1996). Producersand consultantsrefuse to handlesticky materialsand therefore do not like to use sticky traps(G. Wilde, personalcommunication). Accuracy of visual inspectionsof plants may vary becauseof differencesin the experienceof the scout,weather conditions, and the time of day plants are inspected. Plant kairomones have been studied extensively for their attractivenessto com rootwormadults (Jobnson et al. 1985,Lampman and Metcalf 1987,Metcalf and Metcalf 1992,-Hammack1997). Plant kairomonesin a non-sticky trap have potential for being a user-friendly,species-specific tool for monitoringadult westerncorn rootworm.The trap can be usedto detectfirst emergence,insect presence and distribution,and relationshipto pestdamage (economic threshold). A new commerciallyavailable trap manufacturedby TRECE@,Inc. (Salinas,CA) has a clear-plastic,dome-shaped top (5.5cm deep x 8cm diameter)and clear-plastic, cylindrical,removable bottom (6cm deepx 7cm diameter)(Fig. 1). A stun pill bait of 167 cucurbitacinpowder, carbaryl, and an ediblewax cauier is placedinside the domeat the top of the trap and a kairomonelure is suspendedon the outerrim of the domeat thetop of the trap.Beetles are attractedby the extemallure to the trap and encounterthe bait inside the trap. Cucurbitacinis an arrestantand feedingstimulant, so when beetlesencounter the compound,they begin feedinguncontrollably (Metcalf 1987),die and are retainedin the removablebottom of the trap. The Csalomon@trap (Fig. 2) was developed recently by the Plant Protection Institute at the HungarianAcademy of Science(Budapest Hovmann O. 15, H-1022, Hungary). It is of a different design and can be baited with a sex lure, kairomonelure, or both. Beetles that are attracted to and enter the trap are killed by pesticide ships [dichlorovinyldimethyl phosphate(DDVP)] inside the upper and lower comparfrnentsof thetrap (Baca et al.2003). FIG. l. Pherocon@CRwtoup FIG. 2. Csalo-on@ co- rootworm trap. Whitworth et al. (2002) suggestedthe Pherocon@CRW trap may provide a more accurateassessment than visual countsofcom rootwormadults. However,before the trap can be available commercially,factors related to its use need to be determined.The purposesof this study were to: (l) determinethe relationshipbetween the abundanceof beetlescaught in the traps and subsequentdamage by larvaeto corn roots the following year,(2) detenninethe relationship between abundance ofbeetles trapped on the interiorvs. ixterior (outsiderows) of a comfield, and (3) comparethe efficiency of the Pherocon@ CRW trapto monitorthe distributionof westerncom rootwormwith that of the Csalomon@ trapbeing used in someareas in Europe(Baca et al. 2003). MATERTALSAND METHODS Abundance of corn rootworms in 18 fields of com was monitored with the Pherocon@CRWtrap during the growingseasons of 1998-2001.Twelve traps were placed in eachfield and changedweekly. Insects trapped were placed in 17 x 20cm Ziploc (S.C. Johnson& Son, Inc., Racine,WI 53403)bags, taken to the laboratory,and westerncom rootwormbeetles were countcd.Traps were initially placedin a field when plantsbegan silking andmonitored for six weeksor until beetlesdecreased in abundance.The following season,12 plants were randomlychosen and removedfrom eachfield, washed,gdl the amountof damageto the rootsassessed using a standardroot ratingscale of 1-6 ' (Hills and 168 Petersl97l). A simplelinear regression was usedto characterizethe relationshipbetween abundanceof beetles (most countedduring one week) and damagethe following year. A root rating averageof 3.0 or less was consideredan acceptableamount of damagenot resultingin economicloss. Only datafrom fields not treatedfor beetleswere usedin the regressionanalysis. However, data from severaltreated fields were evaluatedin a separate analysis.In addition,date of first beetleemergence in emergencecapes and in the lure trap in the samefield was comparedin at leastone field during eachof the growing seasons (1998-2001). In 2000,Pherocon@CRW traps were placed on eachofthe exteriorrows (24 traps) and interior(eight traps) of t I fields nearScandia, KS. Trapswere changed weekly during the season(l July - 15 August). A simple linear regressionwas used to determinethe relationshipbetween abundance ofbeetles caught at theexterior and interior ofthe field. In 2001,ttaps wereplaced at four distances(0, 3, 10, and 40 rows) into eight corn fields. Therewere four replications(edges) of the field, and trapswere placed30m apart. Numbersof beetlesfapped in eachkind of trap were comparedfor eachdistance each week. Preliminaryanalysis suggested an interactionbetween abundance of beetlesand distanceinto the field. Therefore,the final ANOVA analysisconsisted of comparisons between distancesof traps into the field when numbersof beetles trapped were in three categoriesof< 150,150-300, and > 300per week. Two plant allelochemic-basedtraps (PheroconoCRW and Csalomon$ were comparedfor their efficiency in monitoring abundanceof western, northern (Diabrotica Iongicornis barberi Smith and Lawrence) and southern com rootwom (Diabrotica undecimpunctatahoward, Barber) beetles near Abilene, KS, in 2001. Eight trapsof each kind were placedaltemately on the perimeter(outside row) and inside of six com fields. Traps were at least 30m apart and examinedevery sevendays. Traps were initially placed in the field when com was in the green-silkstage and examinedduring a three-weekperiod. Insectshapped were placedin resealableplastic bags,taken to a laboratory,and the number of beetlesof each species(westem, northem, and southemcorn rootworms) was counted. The Pherocon- cRw trap was baited with a kairomone lure sold by TRECE@and the Csalomon@trap was baited with a kairomonelure sold by the Hungarian Plant Protection Institute. In 2002, twenty-four traps of each kind (Csalomon@and pherocon@CRW; rv"r" placedinside four corn fields.The samemethodology was used as in 2001except both traps were baitedwith sex pheromone,a kairomone,or both, which were purchasedfrom the HungarianPlant Protection Institute. Data were transformed[n (x+l)] to stabilizevariance before statisticalanalysis. Non transfomredmeans are presented in thetables. RESULTSAND DISCUSSION The relationshipbetween abundance ofbeetles one year and scoresofroot damage the following year are shown in Fig. 3. It seemsthat when beetle abundancedoes not exceed200 per trap per week,economic damage would not occurthe following year.More dataare needed on the effectof more than20Q beetles per trapper weekto determineif this relationshipis linear when beetlesare very abundant.Data from the l0 fields infestedby morethan 200 beetlesper plant and treatedwith an insecticidethe weekthe thresholdwas exceededare presented in Table l. In all cases,ratings of damageto rootswere less than the economicthreshold of 3.0. r69 o, .i; _.f.-..'/' GI Y'0 00301'1'1814 v. - R'=07288 o o t 0 200 400 600 800 1000 1200 1400 WCRI/TraPMeek FIG. 3. Relationshipbetween numbers of westerncom rootwolrn beetlescaught in Pherocon@CRW trap andsubsequentrootrating, Kansas, 1998-2000' TABLE 1. Scoresof RootDamage Following Adult CornRootworm Beetle Control in CornFields the PreviousYear, Scandia, KS' . Avg' Root Rating the eek FollowingYear I 305 2.3 2 525 1.8 3 r78 1.8 4 160 2.3 5 r64 2.0 6 278 2.1 262 1.8 8 t67 2.4 9 212 2.0 l0 296 2.1 The first beetlewas caughtin a lure trap tbreedays before a beetlewas detectedin an emersencecase in threeofihe four yearsand on the sameday in one year (Table2). because"thelure irap was attractiveto beetles,using this methodwould be an effectiveway to detect ,*"rg"rr"i of the first beetle. Establishingthis date would be importantin determiningschedules for samplingand treatmentof adults' TABLE 2.Datethe First WestemCorn Rootworm Beetle Was Foundin Emergenceand Tr6c6@Pherocon@ CRw l 998-2001. Year Lure 1998 29 Jun 26 Jun 1999 25 Jun 22tun 2000 22Jw 22hrr 2001 02 Jul 29 Jun and The relationshipbetween the numbersofbeetles caughtin trapsatthc interior suggesttng exteriorof corn fields in 2000is shownin Fig. 4. The R-squarevalue was 0.81, 170 that 8l%oof the variationcould be explainedby trapson the exterior(outside) row of the field. 600 'trxo- y=0.8512x+63.781 500 EE R2= 0.8072 I i= 400 FE YE300 >F ao :lt g 200 g.] 100 = 0 0 100 200 300 400 500 600 Mean# WCR/ExteriorTrapMeek FIG. 4. Comparisonof numbersof western corn rootworm beetles caught with traps on interior and exterior of field. Analysis of the numbersof bestlescaught in traps at different distancesinto the field in 2001 is shownin Table3. when fewer than 1506eetles were caughtper trap,in a given week, significantlyfewer beetleswere in the ffaps in the 0 (outsidi) and third row into the field than in traps l0 and 40 rows into the field (F: g.39, df = 3, p < 0.0001). However,when 150 - 300 beetleswere caughtper trap per week, a range that includesthe ggonomicthreshold (F = 1.63,df = 3, p = 0.20),or more than 300 per week (F = 0.41, = = df 3, P 0'23) therewere no significantdifferences in numbersof bietles capturedat the differentdistances. The resultsobtained in 2000 and 2001 showedthat placingkairomone ffapson the perimeterof a field was an acceptablemonitoring technique when abundance ofbeetlesexceeds the economicthreshold. TABLE 3. Number of Beetlescaught in TRECE@pherocon@ cRW Traps at Different Distancesinto a Field.200l.Field.2001. No./Trao/Weeku Row 0-r50 I 50-300 > 300 0 58.0b 162.7b 488.6a 3 68.6b l82.5ab 534.5a l0 83.7a 2Q1.6a 505.0a 40 6)- la l91.7ab 500.0a Means followed by same letter within a column are no slgnr different (P > 0.05,LSD test). combined data from the six fields for each of the three sampleperiods are summarizedin Table4. Significantlymore western, southem, and northemcorn roofworm beetleswere caught in the Pherocon@cRW thanin the csalomon@trap on mostdates. Two to five time morewestern corn rootwormbeetles were caughtin the pherocon@cRW trap thanin the Csalornon@trap on mostoccasions. Differences were gr€at€r in trapsin the field thanthose on the perimeterof the field. Similarresults were recorded for thjsouthem and northern species.In general,more beetleswere caught in pherocon@cRW traps in a field 171 thanon the field perimeter.These results are similar to thoseobtained in the trapplacement studieswhen beetleswere scarceqs describedpreviously. Approximately equalnumbers of beetleswere caughtin Csalomonotraps insidi and at ihe p.ri*.t.t of a field. Pherocon@ CRWtraps could be examinedand emptied more rapidly than Csalomon* traps. TABLE 4. Comparisonof Com RootwormsCatch by Csalomon@and PherocontCRW Traos.Abilene. KS. 2001. No. WCR No. SCR No. NCR Date Location" Meanb+ SEM Meanb+ SEM Meanb+sEM 6 Aug In Csalomon 6.4+ 1.5a 6.7+ 1.6a 0.7+0.2a pherocon@cRW 33.3+ 8.5b 27.3 + 3.9b 3.2*0.6b Out Csalomon@ 9.3+6.9 ^ 6.5+2.7 a 1.7+ 1.0a Pherocon@CRW 10.6+2.7a 19.4t4.3b 5.3* 1.5b 15Aug In Csalomon@ 6.5+ 1.7a 5.6+ 1.3a 0.6+Q.2 a Pherocon@CRW 2O.O*5.4b 14.3+2.6b 1.2+0.3a Out Csalomon@ 5.3+2.3 a 5.9r 1.9a 1.7+0.7 a Pherocon@cRW 8.5+ 2.5b lo.7 + 1.9a l.l + 0.3a 2l Aug In Csalomon@ 1.6+0.4a 0.6*0.2a 0.03+ 0.03a Pherocon@cRW 5.7+ 1.7a 1.8+ 0.3a 0.5+ 0.1a Out Csalomon@ 0.6+ 0.3a 0.8+ 0.3a Q.3+Q.2 a Pherocon@CRW 3.3+ l.l a 2.7+0.5 a 0.6+0.2 a u In = taps 100 paces into the field; Out : traps around the edge of field. o Means followed bv the same letter within a date and location in a column are not significantlydifferent (P > 0.05). Four to five times more beetleswere caught in PheroconoCRW traps baited with kairomoneand sex lure as in Csalomon@traps when beetleswere either abundant(Scandia) or scarce(Abilene) (Table 5). When trapswere baitedwith only the kairomone,similar resultswere obtained (Table 6). Whenboth trapswere baited only with the sexlure, similar numbersof beetleswere caught in both traps(Table 7)' TABLE 5. Number of WestemCorn RootwormBeetles in Csalomon@and Pherocon@ CRW Traps with Kairomoneand Sex Lure, Scandiaan9 Abilene, KS, 2002' Date MeanuNo. MeanoNo. Csalmon@ PheroconoCRW P-value Scandia,KS I t75.79b 619.37a < 0.0001 2 165.29b 533.43a < 0.0001 J 183.39b 383.52a < 0.0001 Combined t74.67b 499.10a < 0.0001 Abilene,KS | 3.22b 11.40a < 0.0001 2 1.70b 6.32a < 0.0001 Combined 2.34b 8.49a < 0.0001 row by location are not sigtificantly different' (P> 0.05). 172 TABLE 6. Number of WesternCom RootwormBeetles in Csalomon@and Pherocon@ CRW Trapswith KairomoneLure, Abilene, KS,2002. Trap Mean"No. P Csalomon 1.28a 0.0024 Pherocon@CRW 6.52b 0.0024 Meansfollowed by the sameletter within a row arenot significantlydifferent (P0.05). TABLE 7. Number of WestemCom RootwormBeetles in Csalomon@and Pherocon@ CnW fr"ps witl Sexfure, Vlm Date Mean"No. MeantNo. Csalmon@ Pherocon@CRW P I 8.ll a 7.52a 0.64 2 7.30a 7.23a 0.95 3 5.70a 3.77a 0.08 Combined 6.70a 5.90a 0.15 Means followed bv the same letter within a row are not signifi cantly different (P> 0.05). Results of these tests suggestedthat Pherocon@CRW traps can be used for monitoring abundanceof corn rootworms and making fieatment decisions. Traps are independentofthe experienceofthe individualperforming the surveyand tend to average out the influenceof environmentalconditions. These traps are also more likely to be acceptedby growers/consultantsbecause there is no exposureto sticky substances.Our results suggestedthat an averageof fewer than 200 beetlesper trap during any one week- intervalwill not resultin an economicloss caused by root damagethe following year.More dataon the damageloss relationship are needed when beetles are abundant. Our dataalso suggestedthat abundanceofbeetles caughton the exterior(outside) row ofa corn field usuallyreflected abundance in the field. Therefore,numbers ofbeetles per hap canbe usedto predictwhen abundance of b€etlesis nearor exceedsthe economic threshold.In other studies,we havedetermined the sphereof influenceof the Pherocon@ CRW lure trap attracted beetles from about 100 feet away (G. Wilde, personal communication).This might help explainwhy the samenumbers of beetlesmight be caught in trapson the exterioror outsiderow as inside a field when beetlesare abundant.The numberand placement of trapsneeded in a field arebeing studied. More beetleswere caughtin the Pherocon@CRW trap than the Csalomon@trap no matterwhich lure (TRECE@oicsalomon$ was attachedto ihe trap.The Pherocon'iRW trap was also easierto handleand change.Therefore, we suggestthe useofthe Pherocon@ CRWtrapto monitordistribution and abundance of westerncom rootwormadults. ACKNOWLEDGEMENT This paper is contributionNo. 04-081-Jof the KansasAgriculture Experiment Station. t73 LITERATURE CITED Baca, F., T. Kevesi, R. Sekulic, P. Melnik, O. Bukachuk, and B. Kushniv. 2003. Comparisonof efficiencybetween Ukranian haps and pheromoneCsalomon traps in monitoring Diabrotica virgifera virgifera LeConte occurence in 2002. IWGO Newsletter24:22-23. Chiang,H. C. 1973.Bionomics of the northem and westerncom rootwolm. Ann. Rev. Entomol.l8:.47-72. Hammack,L. 1997. Attractivenessof syntheticcorn volatiles to feral northern and western com rootwormbeetles (Coleoptera: Chrysomelidae). Environ. Entomol. 26:.311'317. Hills, T. M., and D. c. Peters.1971. A methodof evaluatingpest planting insecticide treafinentsfor controlof westemcom rootwormlarvae. J. Econ.Entomol. 64:764-765. Hein, G. L., and J. J. Tollefson. 1984.Comparison of adult com rootwonn (Coleoptera: Chrysomelidae)trapping techniquesas populationestimators. Environ. Entomol. 13: 266-271. Hofftnann,M. P., J. J. Kirkwyland, R. F. Smith, and R. F. Long. 1996.Field testswith kairomone-baitedtraps for cucumberbeetles and corn rootwormsin cucurbits.Environ. Enotmol.25: ll73-ll8l. Johnson,T. B., D. R. Raemisch,A. s. Las, andF. T. Turpin. 1985.Response of westenn com rootwonn (Coleoptera:Chrysomelidae) adults to several semiochemicalsin comfields.J. Econ.Entomol. 78: 85-88. Lampman,R. L., and R. L. Metcalf. 1987Multicomponent kairomone lures for southem and western com rootworms (Coleoptera:Chrysomelidae: Diabrotica spp.). J. Econ. Enotomol.80: ll37 -1142. Luckmann,W. H., J. T. Shaw,D. E. Kuhlman,R. Randell,and C. D. Lesar.1975' Corn rootwormpest management in canningsweet com. Ill. Nat. Hist. Surv.Circ' 54' Metcalf.R. L. 1987.Plant volatiles as insect attractants. CRC Critical Reviewsin PlantSci. 5:251-301. Metcalf,R. L., and E. R. Metcalf. 1992.Plant Kairomones in InsectEcology and Control. Chapmanand Hall, New York.. Musick,G. J., andM. L. Fairchild.1970. Field emergencecage for cornfootworm adults. J. Econ.Entomol. 63: 17 10-17 ll. Sharv,J. T., W. G. Ruesink, S. P. Briggs, and W. H. Luckmann. 1984' Monitonng populationsof corn rootwormbeetles (Coleoptera: Chrysomelidae) with a trap baited with cucurbitacins.J. Econ.Entomol. 77:1495'1499. Whitworth, R. J., G. E. Wilde, R. A. Shufran,and G. A. Milliken. 2002. Comparisonof adult corn rootworm (Coleoptera:Chrysomelidae) sampling methods. J. Econ. Entomol' 95:96-105. t74 SEPT.2OO4 vol..29 NO.3 SOUTFTWESTERNENTOMOLOGIST DIFFERENTIAL MORTALITY OF NATURAL ENEMIES E)(POSEDTO AVOCADO LEAVES TREATED WITH MALATHION BAIT SPRAY DURING A MEDITERRANEA}.I FRUIT FLY ERADICATION PROGRAM W. L. Yeer andP. A. PhilliPs cooperative Extension,University of california 669 county SquareDrive, suite 100' Ventura,CA 93003,U.S'A. ABSTRACT Detection of the Medite,lraneanfruit fly, Ceratitis capitata (Wiedemann)'in Ventura Connty, California" U.S.A., in 1994triggered an eradicationprogram that involved aerial malathion bait (com synrp) spraysapplied on avocadoorchards. The impactsof the sprays on representativeUenefrCiat ipecies during the entire eradicationprogram were studied. The abnndanceof phytoseiid mites, Eusianshibisci (Chant), in the field was not affected by the bait sprays. Laboratory bioassap of the parasitoid Aphytis melinus DeBach exposedto leavessprayed during the progarn indicatedit was highly susceptibleto spray t".ido"., especially on exposed, outer canopy leaves. Bioassays of lanral and adult lacewinp, Chrysoperlarufilabris Burmeister,exposed to leavesindicated larvae were less susceptiblethan adults. Adults were moderatelysusceptible to residueson outer canopy but not on innet canopy leaves. The results suggestthe spray program causeddifferences in mortality of natural enemies and their life stagesand that malathion residue levels affecting thesedifferences were depende'lrton canopylocation. INTRODUCTION Detrimental side effects on natural enemy speciesand other non-target arthropods causedby malathion bait sprays targeted against the Mediterraneanfruit fly, Ceratitis capitata jwieoemannl (Medfly), are well documented. A detailed assessmentof g malathion bait spray progam for C. capitata from 1980-1982in the San FranciscoBay are4 California" inAcatiC that many non-targ€t organisms were negatively affected (Dahlstenet al. 1985). Theseincluded natural enemiesin the ice plant system(washbum .t ut. tlAt;, a variety ofpollphagous andcamivorous arthropods (Troetschler 1983)' gall midge parasitoids(dhler et at. tiS+;, linden and tuliptree aphid parasitoids(Daane et al. l99d), and chalcidoidparasitoids ofwhiteflies (Hoelner and Dahlsten1993). ln Israel, the scale insect parasitoid Aphytis holoxanthus DeBach was highly susceptible to malathion-lacedbait (Cohen et at. teAZ;. In Australia, malathion residues(with oil) on citnrs foliage were highly toxic to the Califomia r€d scale parasitoid Aphytis rnelinw DeBach tor rc-Zg aays after application (Campbell 1975). Other studies on parasitoids (Bartlett 1963,Abdeliatrman 1973, Wilkinson et al. 1975)exposed to malathion(without b"ig *o" consistent with these results. Evidence exists that destruction of natural enemiesdruingMedfly eradicationprogramscan lead to host outbreaks(Ehler et al. l9M)' ffiDepartnent of Agriculture, Agricultural Research Service,Yakima Agricultqral ResearchLaboratory 5230 Konnowac PassRoad, Wapato, wA9895l,U.S.A. 175 th. major - -.r citrus- and aygcado-growingregions of ventura county in southern califomia, insect management historicily mi reiied heavily 6;; l;#*y-reared or naturally-occurdngbiological conrrolagents (uSDA 1999). In.iil;, ;;";;t"ids suchas Aphytis. tp-p rd Metaphycas helveorw (comp.) and various preaaioi, t urre tong uee., recognizedas importantagents in managingpeiti lo.nach l95i). tn "uo""ao, nai'ally: occurringbe.ne!9ial species such as thJ pfiytoseiidmite Eusiei hiJ;r"i lCi-tl, anystio mites, coccinellids such w Stethorusji"pr, (Casey), Hippodanii spp., various parasitoids such as Encars-iaor Erehnocirus spp., the i"""*itii chrysoperla rufilabris Burmeister, and spiders also are presumedto'iray a role in ir-"iini pests such as tglranrchid(spider) mites,amorbia moths, whiteflies, mealybugs, and-sca]e'insects. For !!is- reasorr,many growers and pest contot advisors were concemed over the fate of biological control agents g! integrated pest managementprograms as a large Medfly eradication program was initiated in ociober 1994 in ventura' county. trJ potentia impact ofthe residual malathion bait sprayson avocadoleaves on biological control agents was ofconcern becausethere had beenno history ofextensivemalathion use on avocado in this county. The aim of this study was to determinethe potential impacts of the malathion bait spray used in the 1994-1995Medfly eradicationprogram in ventura county on representative'beneficialspecies, specifically E. hibisci in the field and,A. melinusmd C. rufilabris in the laboratoryusing avocadoleafbioassays. Aphytis melinuswas chosenas a representativeparasitoid becauseit was commercially available for bioassaysthroughout the eradicationprogram. The susceptibility of both larval and adult C. ruplabris exfosed to malathion residues on avocado leaves resulting from the spray progftun was determined. MATERIALS AND METHODS After two mated c. capitata females were trapped in ventura county in late September 1994, a Medfly quarantine boundary was established in the county, encompassingapproximately 225 lfir' and parts of camarillo, Somis, Moorpark, and Thousand oaks. An additional 56 c. capitata males were trapped in camarillo by 7 october. Thesedetections triggered a 42 1 176 Leaves for bioassays(separate residue tests after application dates)using A. melinus I or were collected on eight datis hom 17 Januaryto 9 May 1995. Collectionswere made ground 2 days after spray af,plications. Ten leaveswere randomly selected1.5-2 m-above from the outer anilinner canopies from each tree. Five trees within each of the thiee the orchard sections over an elevation gradient of 20 m were sampled. Leaves from different sectionswere consideredseparate replicates. Becauseaerial sprays,especially at rather than a relatively high elevation, tend to ioncentrate spray depositso4o !h: outer inner canoiy lJaves,outer and inner canopyleaves were comparedin laboratorybioassays' Adulis of ,4. melinus were obtainedfrom an insectary(Fillmore lnsectary,Fillmore' califomia) and were no more than I day old. Bioassayswere conductedby exposingl0 individuals per replicate in ventilated 40-dramvials to a single leaffrom either the outer or inner canopy in the treatedorchard, or from just the outer canopyin the u-ntreatedorchard' There weri-l0 replicates (= number of leavis) for the untreatedleaves, five fiom eachof two orchard sectionsin the relatively flat control orchard,and 15 replicatesfor inner and outer treated leaves, five from each of the three orchaxdsections in the steeper'treated orchard.Water but no food was provided. Test room temp€raturewas 23.9oCand ambient humidity approximately 75Yo. iercutage mortality readingswere made at I day md at 2 or 3 daysofexposure. Laf s*tpting for C. ru/ilabris bioassayswas the same as for A. melinus, except leaves for bioassays were collected on 14 dates from ll October 1994 (one day preheahnent) to i5 April 1995. An insectary-reared, susc€ptible strain @ue'na biosysterns,'Ventura Califomia'1of C. rulilabris wa5 used in bioassays' Eight seconG and third-instar larvae, and eigit adults per replicate were exposed in ventilated Petri dishes (9-cm diameter) to an outer or inner leaf from each of the three sectionswithin untreatedand treated orchards. There were tbree replicates(= number ofeaves) for each ofthe following four treatnents: l) outer leaves,larvae, 2) inner leaves,larvae, 3) outer leaves,adults, and 4) inner leaves,adults. Lacewingswere provided water, but no food, in all but the first two bioassays. Test room conditions were 26.7-29,9oCand 80olorelative humidity. High control mortality occrured in the first bioassaybecause water was not provided, so water was addedin all subsequentbioassays. Percentagemortality readings were madea I day and 3 daysofexposure' Mortality data were subjected to angular transformation and one-way analysis ot variance(AI.iOVA). Sigrrificantresults were followed by mean separationusing Duncan's New Multiple Rangetest (P< 0.05)(SAS Institute 2001). RESTILTS There was no obsefl/edimpact of the malathion bait sprayson E. hibisci at any time during the eradicationprogr"-. In fact, E. hibisci populationswere consistentlyhigher in the treated than unheated orchard (Fig. l). Numbers in both orchards increasedfrom March through May, even after nine malathion bait spray applicationshad bqen made in the treatedorchard. Bioassaysof A. melinus on inner and outer canopy leaves from the treatedorchard comparedwith outer canopy leaves from the untreatedorchard prod-ucedvery sfriking differences. On eight of nine bioassaysat I day of exposure,mortality was highest on treatedouter leaves. On five of six bioissays at 2 or 3 daysof exposure,mortality was also highest on treated outer leaves. Exposureto treated inner leavesresulted in intermediate mortality that was often sigrrificantly greaterthan the confols (Table l)' kr iontrast to A. meliius, larvai C. ru/ilabris exposedto treatedouter leavesfor I and 3 days showed significant mortality on only two and three out of 13 post-spray bioassays,respecuvEly (Table 2). Larval mortalfy increasedover time within a given bioassaybecause of siarvation as larvae were purposely not provided with food. When 177 qt ,H I 0) g q) Fl !D ..o55 IE €r6l\orf)\o t^ Eu +t +t +l +l +t +l +l IY 6 O\OF(a-o\ !)C (frc\O\stt-r \o* F IE c) t.9 to IH l(E to IC' 6stct(iRl t> k (\dr+ tfit q) lo +t +t +t +t +t +l +l FO\O\@O\h a to o\ o\ o\ o\ c ct\ o\ l(t lg o tr q) x BF ! q os! r lrl ('l€-$t-$ E6 = q o >| tu +l +l +t +t +t +t +l i ! o\o\Fri o r)O N-f O\d\Oh h = (q 4i o C) 6 .() (\ o E o x G) o XE i5q) €€-€€€€€8 o0 ad 9tr +t +t +l +t +t +t +t +t +l +l 6 €o\o\HC"tdt(vrF- o 0 EV; F 61 6lr"rN9FNcn6l cq !) =o\ 6 .= o\ CL 6- (t o -o\E+ () 6 .g o\ () o =.-hc o g6 g,Sro$SS€Sg \o a {f tl X :! =l +l +l+l +t+t +t +t +t +l +l o ^= _tro O\OOc4olO\r/)-N tr rr: I F \o\o$Fo\o\@o\F t- aO € +t o o ; o a xE o -tl o o :A e ai o hbo b< o 9: iBq)tr lcg -Eis.3.is {) i:6 >E Rio | o6 i +l+l+t+t+l+l+l +l () g.EEF b0!) tu !oroo\91 f'.tri* (\ a.i gr3 im 9!t a>,;E o d5'AF o &a o> g f;r >\a dH x 'i; cl tt o E q'l ktd c) E9 o= Nr o SU f€ (0 SO ct F ' ..;tr 3 SEEE rrr ,Y dat FtsSE qq !r a) 6A s {5 6F EpnSEe5eEn a(l J€ F- F-;s;-.aa E€ -H oco FA rgF -t\--61 -6lO\EaA s t78 q F o I g !f-$f" rf o0 6 () S*€g,ot-to-t i lt+t+l+t+t+t+l+l+li *,*'*' +l o c) g (\ tro.t9\o€6ttO- iN 179 MalathionBait Spray Dates 1994-1995 1U12 10t24 11t1411t30 1,,1312128 1!'17 47 ?i15 A14 \tn 4A1 M25 5/9 5/23 ,I, J J .I, ,t ,t ,t ,l,J ,1,.1, ,f .1, ,1,,1, E o lzo o_ ffo +l )lc € i,+ .9 3s uj o^ zz c o E1 3/15330 4t134ti' 5t11st24 6n sampe oltes FIG. l. Numbers of Eusieushibisci in untreatedand freatedavocado orchards during the 1994-1995malathion bait spray program againstthe Mediterraneanfruit fly in Camarillo, California. averagedacross all bioassays,lower larval mortality was seen on untreatedand treated inner leaves and untreatedouter leaves than on treated outer leaves at I and 3 days of exposure(Table 2). Adult C. rufilabris exposedto heat€d outer leavesat I day and 3 days of exposure showed sigrificant mortality in six and four out of 14 post-spraybioassays, respectively (Table 3). Significantmortality was associatedwith leavesthat showedvisible bait spray deposition. Visibly, outer leaveshad greaterbait sprayresidues than did inner leaves. Adult lacewing health and mortality varied considerablyfrom bioassayto bioassay(Table 3). Elevatedtemperatures within the insectarybioassay room causedhigh mortality across treatmentsin two bioassays(after 15 Februaryand 11 April applications).When averaged acrossall bioassays,adult mortality (Table 3) was 3.6 times higher than larval mortality (Table 2) on treatedouter leavesat I day ofexposure. DISCUSSION The 1994-1995 malathion bait spray prognm against the Medfly in southern Califomia seemedto have no impact on E. hibisci, which is highly significant becausethis mite is the most abundantgeneralist predator in avocadoorchards (McMurtry and Johnson 1965).kt addition to tehanychid mites, it prela on the thrips Scirtothripsperseae Nakahara (Faberand Phillips 1998),the major insectpest ofavocado (Yee et al. 2001).Treated E liDnci populations increased in numbers despite repeated exposures to malathion, suggesting these mites are quite tolerant of this insecticide. Mite populations 180 o .+r-SSSogoS,a€i.+g c) = @ € +t +t +t +t +t +t +t +t +t +l +t +t +t +t +t +l I -(rr'arN{raci-6lr o0 ! \o co\o-h 6 '6 o El- \o F\6tGt-ht\t-sf, -o\--o\al sf >l -l a6l o c(.)l EIJI Cd a-O I ^=6^ () 3lElu r 9r*sFr-,- *od*It.'o gl-'E t+tl +l +l +t +tE +t +t +t +t +l +t +l +t x orl JJ o\ooiN €<'--st-o r\ ioi l8t typically increasein spring,--coincidingwith avocadoflowering andlroduction ofpollen on whjch mites feed (McMurtry and Johnson 1965). This population increasewas also seenduring the spray program, further suggestingthe mites were unaffectedby malathion bait sprays. Eusieus hibisci also feeds on mealybug honeydew (McMurtry and scriven 1964),but results suggestit did not feed on the corn synrp bait or, ifit did, was unaffected by the malathion. Also, because of its negatively phototropic behavior, the mite is normally found on undersidesof leaves, where its exposureto malathion was probably minimized. Insecticideresistance occurs in E. hibisci (Hoy 1985),but whetherresistance occurredin the study population was unknown. organophosphateswere not known to be usedin the avocadoorchard or surroundingareas. The high mortality of A. melinuscaused by bait sprayresidues was consistentwith studiesof A. melinus (Campbell 1975),A. holoxanthus(Cohe;n et al. 1987),and other parasitoids@hler et al. 1984, Daane et al. 1990). In addition, the current results suggest parasitoidsthat frequent the more protectedinner canopy may suffer lower mortality than those that frequent the outer canopy. The effects of malathion bait sprays onnon-Aphytis parasitoidsin avocadoorchards [e.g., the eulophid Ceranisusmenes (Walker\ (Faber and Phillips 1998)l under different canopy conditions need to be assessedin future spray programs. Comparedwith l. melinus, the malathion bait spray residuescaused relatively low larval and adult C. ruJilabris mortality, especially when the lacewings were exposedto inner leaves. The lacewing Chrysopasp. was less susceptibleto certain organophosphates thaurNabis sp. and coccinellids(van den Boschet al. 1956),and insecticideresistance has been detectedin C. carnea @ree et al. 1989, Grafton-Cardwelland Hoy 1985). This information and our results suggest lacewings can rebound more quickly than some parasitoidsliom anypopulation suppressions caused by malathionbait sprays. The lower mortality of C. rufilabris lawae comparedwith that of adults exposedto both outer and inner treated leaves probably resulted from stage differences in feeding biology or physiology. Larvae,being predaceousonly, probably did not feed on the malathion-synrpresidues on the bioassayleaves; adults, being sugarfeeders (Hagen et al. 1970),probably did. Physiological differencesin insecticidedetoxiffing mechanismsmay exist betweenstages. Larvae of C. carnea are also less susceptibleto malathion and other insecticideresidues than adults (Bartlett 1964, Wilkinson et al. 1975,Grafton-Cardwell andHoy 1985). In conclusion, the results suggest the 1994-1995 bait spray program caused differencesin the mortality of natural enemiesand their life stagesin avocadoorchards. ln addition, they suggest that malathion residue levels affecting these differences were dependenton canopy location. Thesedifferences need to be consideredwhen evaluating the full impact of malathionbait sprayson pest management. ACKNOWLEDGMENT We thank Lynette Buchanan-Rothand Jim Lloyd Butler for allowing us to use tleir avocadoorchards, and Sue Mills, Jan Dykes, Chris Roth, and John Rodgersfor field and laboratory assistance. We also thank Jake Blehm and Alvaro Sequiera of Buena Biosystems Insectary for fumishing assistance,space, and lacewings and Fillmore Insectaryfor supplyingthe parasitoids. LITERATURECITED Abdelrahman,l. 1973. Toxicity of malathionto the naturalenemies of Califomia red scale,Aonidiella aurantii (Mask.) (Hemiptera:Diaspididae). Austr. J. Agric. Res' 24: ll9-133. 182 Bartlett, B. R. 1963. The contact toxicity of some pesticideresiduest9 hperrupterous parasitesand coccinellid predators' J. Econ. Entomol' 56" 694-698' Bartled B. R. 1964. Toxicity of rom" pesticidesto eggs,larvae, and adults of the green lacewing,Chrysopa carrrea' J. Econ.Entomol. 57:366-369' and spray oil on Campbell,' fvf. frf. f iZS. Duration of toxicity of residuesof malathion citrus foliage in south Australia to adults ofa california red scaleparasite Aphytis melinus DJBach (Hymenoptera:Aphelinidae). J. Aust. Entomol. Soc. 14:.16l- 164. cohen, E., H. Podoler,and M. El-Hamlauwi. 1987. Effectsof the malathion-baitmixture used on citrus to control Ceratitis capitata (Wiedemarur)(Diptera: Tephritidae)on the Florida red scale,chrysomphalus aonidium (L.) (Hemiptera:Diaspididae), and its parasitoid Aphytis holoxaithus DeBach (Hymenoptbra: Aphelinidae). Bull. Entomol.Res. 77: 303-307. fruit Daane,' K. M., D. L. Dahlsten,and S. H. Dreistadt. 1990. Effectsof Mediterranean fly malathion bait spray on longevity and oviposition of parasitoids-oflinden and tttipnee aphids(Homoptera: Aphididae). Bnviron. Bntomol. 19: 1130-1134. Dahlsten,O. 1., f. B. Hoy, D. L. Rowney,K' A' Hoelmer,M. Wilson,K' M' Daane,W' A' Cooper,D. C. Weber, L. E. Caltagirone,D. J. Clair, and S' Marcandier' 1985' Effeits of malathion bait spray for Meditenaneanfruit fly on non-targetorganisms on urban trees in northem Califomia. University of Califomia, Berkeley' Final progressreport to Califomia Departmentof Food and Agriculture. Agreementno. 1781. DeBac[ P. 1951. The necessityfor an ecologicalapproach to pest confol on citrus in California. J. Econ.Entomol. 44l.443'M7. Ehler, L. E., Endicott, M. B. Hertlein, and B. Alvarado-Rodriquez. 1984' Medfly eradicationin California: impact of malathion-baitsprays on an endemicgall midge and its parasitoids. EntomologiaExpt. et Applicata. 362201-208. Faber,B. A., and P. A. Phillips. 1998. Avocado thrips subproject2: pesticideevaluations and phenology-Califomia in thi field, pp. 2l-22. In 1998 Califomia Avocado Research Symfosium. Avocado Society andUniversity of California, Riverside. Grafton-Cardwell, E., and M. A. Hoy. 1985. Intraspecific variability in responseto pesticides in the common green lacewing, Chrysoperla carnea (Stephers) (Neuroptera:Chrysopidae). Hilgardia. 53: l-31. Hagen,K. S., E. F. Sawall,Jr., andR. L. Tassan.1970. The useof food spraysto increase effectivenessof entomophagousinsects. Proc. Tall Timbers Conf' Ecol. Animal ControlHabitat Management. l: 59-81' Hoelmer,K. A., and D. L. Dahlsten. 1993. Effectsof malathionbait sprayon Aleyrodes spiraeoides (Homoptera: Aleyrodidae) and its parasitoids in northern Califomia. Environ.Entomol. 22: 49-56. Hoy, M. A. 1985. Recent advances in genetics and genetic improvement of the phytoseiidae.Annu. Rev' Entomol. 30: 345'37O. McMuriry, J. A., and G. T. Scriven. 1964. Studieson the feeding,reproduction, and divelopment of Amblyseius hibisci (Acarina: Phytoseiidae)on various food substances.Ann. Entomol.Soc. Amer. 57:649'655. McMurtry, J. A., and H. G. Johnson. 1965. Some factors affecting the abundanceof the pi"du""our mite Arnblyseiushibisci in southemCalifomia (Acarina: Phytoseiidae). Ann.Entomol. Soc. Amer. 58:49-56. Pree,D. J., D. E. Archibald,and R. K. Morrison. 1989.Resistance to insecticidesin the common green lacewing chrysoperla carnea (Neuroptera: chrysopidae) in southemOntario. J. Econ.Entomol. 82:29-34' SAS Institute. 2001. SAS/STATuser's guide, version 8. Cary'N.C. 183 Troetschler,R. G. 1983. Effectson nontargetarthropods of malathionbait spraysused in California to eradicatethe Meditenaneanfruit fly, Ceratitis capitata (Wiedemann) (Diptera:Tephritidae). Environ. Entomol. 12: lg16-1922. USDA. 1999. Crop profile for avocadosin Califomia. http://cipm.ncsu.edu/cropprofiles/ docs/caavocados.html. van den Bosch,R., H. T. Reynolds,and E. J. Dietrick. 1956. Toxicity of widely used insecticidesto beneficialinsects in Califomia cotton and alfalfa fields. J. Econ. Entomol. 49:359-363. Washburn,J. A., R. L. Tassan,K. Grace,E. Bellis,K. S. Hagen,and G. W. Frankie. 19g3. Effectsof malathionsprays on the ice plant insectsystem. calif. Agric. 37:30-32 wilkinson, J. D., K. D. Biever and c. M. Igaoffo. 1975. contact toxicity of some chemical and biological pesticidesto severalinsect parasitoidsand predators. Entomophaga.20: ll3 -12e. Yee,W. L., P. A. Phillips,J. L. Rodgers,and B. A. Faber. 2001. phenologyof arthropod pestsand associatednatural predators on avocadoleaves, fruit, and in leaflitter in southernCalifornia. Environ.Entomol. 30: 892-898. I tt4 vol.29 NO.3 SOUTHWESTERNENTOMOLOGIST SEPT,2OO4 COMPARATTVEBIOTOGY OF CHINCH BUG, BI.I.9STISLEUCOPTERUS LEUCOPTERUS(SAY)I, IN RICE AND SORGHUM o. I. Mejia-Fonc3,M. o. way' And J. K. Olsona ABSTRACT Developm.e,ntof hatched eggs, first-, second-,third-, and fifih-instar nymphs, and adults of the chinch bug, B&1rsasleucopterus leucopterus (Say), showed no significant differeNrceson rice or sorghumin the laborarory;however, developmental time betweenthe third- and fourth-instars was significantly diffelent for insectsr€ar€d on rice and sorghum. After 75 days, most of the fifttr-instar nymphs had not molted to adults; thefefore, conclusionscould not be draumregardmg differences in developmentof fiffh-instar nymphs to adults on rice and sorghum. Developmentfrom egg to fifth-instar was about 60 days on both hosts. Cephalic capsulewidths of second-,third-, and fourth-instarswere significantly narlower in insectsrear,ed on rice (288 vs 302 p.m for seoond-,358 vs 370 pm for third- and 418 vs. 434 1tmfor fourth-instars). Pre-oviposition time was 7 days eadier for females hrq€d on sorghum than on rice; howwer, the total number of eggs laid per female did not tliffer significantly betweenhosts (50.5 and 52.5 eggslaid per female for rice and sorghum; respectively). Data indicated that sorghwir was a slightly better host for chinch bug than rice, but also indicaed ttre suitability of rice as a host INTRODUCTION The chinch bug, B&sszs leucopterus leucopterus (Say), is a pest of sorghunl Sorghum bicolor (L.) Moench., (Cronholm et al. 1998); com, Zea mays L. @orter a al. 2002); wheat, Triticum aestivumL.; and other small gnins (t€onad 1966). However' not rmtil recently has the chinch bug receivedmuch attentionas a pest of rice. Chinch bugs re commonly observed on rice in various growth stages in Texas; howwer, the most conspicuousdamage is associatedwith death of seedlingsearly in the season. Damageis caused by overwintered adults in paddies and on levees before the permanentflood is established(way et al. 20@). After leaving overwintering sitesin the spring, adult chinch bugs move to a wide rangeof Poaceaehosts including spring and winter wheat; barley, Hordewn vulgarc L.; oats,Avena sdiva L; tye, Secale cereale L-; sorghum; and com (Snelling 1936, Painter 1951); Sudan gas, Sorghwn sudanense(Piper); broom corn, Sorghtmtvulgare L.; Indian and field com; and millet, Pennisetumamericanum (L.) (Flint and Lanimer l926,I.eornaud1966). Adults Lygaeidae 'rDepartnent]Semiptera: of Entomology, TexasA&M Univenity Agricultural Reserch and Exteirsion Ce,nter,1509 Aggie Drive, Beaurnont,TX 77713,USA \ oOepartrrent of Entomology, Texas A&M Universig, 2475 TN/IIJ, College Station, TX 77843,USA 185 mate and femaleslay eggs inside the sheathsof lower leavesand on the roots or crowns of plants growing in loose, cloddy soil. At 30 + 2"c nd 60 + lTyo relative htmidity wittr sorghumas a host, female and male chinch bugs required 36.0 and 31.6 days on average, respectively,to reachthe adult stageafter passingthrough five instars(Vasquez and Sancfez 1991). Underthese conditions, the averageduration for eachof the instarswas 5.5, 3.6,4.5, 1.2, and 7.8 days. overwintered female chinch bugs producedmore eggs and lived longer than did females of the first and second generations(Janes et al. 1935). For second- generationfemales maintained at 24.5oc on wheat, preoviposition time ranged from 8-15 days, oviposition time from 49-63 days, total ntunber of eggs laid from 129-318, and longevityfrom 7l-73 days. Although no rearing conditions were specified, developmentaltime in days (from first-instar to adult) of chinch bugs reared on various grass hosts was reported by Burks (1934) as follows: bermudagrass,Cynodon dactylon (L.) Pers, 33; timothy, Phleumpratense (L.), 35.6; Kentucky bluegrass,Poq pratensis (L.), 35.0; corr; 33.3; meadow foxtail, Alopecuruspratensis (L.), 36.1; chinch bug-resistantsorghum, 36.3; reed canary grass, Phalaris arundinacea(L.), 36.1; Hungarianmillet, Setaria italica (L.),36.2; wild badey, Hordeumjubatum L., 35.2; quack grass,Agropyron repens(L.), 35.5; Washingtoncreeping bent grass, Agrostis palustris Huds., 37.0; little blue.stem grass,Andropogon scoparius Michx., 36.0; and Sudan grass,Sorghum vulgare var. sudanenseHitchc. 34.3. Thesedata show developmentaltime was similar for chinch bugson all hoststested. A number of studies in the literature concem factors determining suitability and preferenceinvolving different Poaceae.ln a four-year study, Benton and Flint (1938) found that preferencefor small grain crops varied accordingto spring weatherand the development ofeach specificcrop. The developmentofchinch bug on different Poaceaehosts is also influencedby the susce.ptibilityofeach specific host to the insect (Leonard 1966). In Kansas,native grasses were rarely damagedby chinch bug, but many introducedgrasses were susceptible(Hayes and Johnson1925). Apparently, perennialbunch glassesbecause of'barsh" tissue support abundantchinch bugs without being damaged.Smith et al. (1981) compareddevelopment of chinch bugs on commonly grown cultivars of wheat (Newton), barley (Reno),com (DeKalb XL 454), and sorghum (NC-170) and found that females laid significantly more eggs on sorghumthan on wheat or com. Also, more eggswere laid on badey than on ufieat or corn. 'among Although perceotageof harchedeggs and longevity of adults did not differ the four hosts, nym,phsdeveloped faster on sorghum than on corn" barley or wheat. Dahms et al. (1936)also reported differences in fecundity,size, and rate ofdevelopment ofnymphs reared on sorghumand other host plants. Worldwide, there are few referencestp rice as a host of chinch bug. Likewise, no previous research has been conducted on chinch bugs in rice in the United States; consequently,basic informatior4 suchas the suitability ofrice as a host for developmentand reproductionofchinch bug, is lacking. Thus, our objective was to study the development and oviposition of chinch bug on rice comparedto that on sorghum. MATERIALS AND METHODS Experiments were conducted in 1996 at the Texas A&M University Agxicultual Researchand Extension Center at Beaumont,TX. Seedsof 'Gulftnont' rice and 'AP9850' sorghum were planted in 9-cm-diameterby 6-cm-deeppots filled with field-collected and sifted League soil. Pots were placed in a greenhouse(30 + 8'C and 70 + l5yo relative humidity). The soil was fertilized at planting with ureaat a rate equalto 128kg N per ha. At emergence,seedlings were thinned to five rice or sorgbum plants per pot. Weeds were removedby hand. Adult chinch bugs were collected from untreatedrice in the TexasRice Belt. 186 'Ihree weeks after emergence,50 pots each of rice and sorghum were individually cagedusing transparentacetate cylinders 7.5-cm in dianeter by 25-cm tall. Each cylinder was ventilated by two screenwindows and a top. Plants in each cagewere infested for 72 nours with eight adult male and eight adult female chinch bugs. After removal of insects, cagedplants were trans ned to a growth chambermainrained at 26.6"C for 13 h of light, 15.5'eCfor I I h of darlg and 55 + 25o/orelalle hurnidity. Plantswere wateredabout every 4 days. These conditions mimiced those in the Texas Rice Belt in April when chinch bugs leave ovetwintering sites and move to young rice. Temperatures and photoperiod programmedinto the growttr chamberwere obtainedfrom lO-year averagesrecorded at the Texas A&M University Agricultural Researchand Extension Center at Beaumont and JefrersonCounty Airport MeteorologicalService, respectively. From onset of egg hatch (12 days after infesting plants in cages)to 75 days later, three to fonr pots of rice and sorghumwere re'movedfrom the growttr chamber every4 to 7 days. Plantsin pots were dissectedand the soil inspectedfor eggs,nymphs, and adultswhich were placed in vials containing75o/o ethyl alcohol. A binocular microscopewas usedto help separatenymphs by instar. During the experiment plants were changedas needed,and a camel'shair brushwas usedto transferinsects to cagedpots of freshrice or sorghum. The number of insects of each stageor instar producedon both hosts over time was compared. Also, the time required for 5oo/oof the individuals of a specific stageor instar to reach the next one was determinedfor eachhost. To oompar€development of nymphs betweenhost species,cephalic capsulewidths of 25 randomly selectedrqdividuals of eachinstar per host werp measuredusing a binocular miooscope fitted with a micrometerin the eyepiece. Chinch bug oviposition was determined using the same materials and methods describedabove. Twenty pots eachofrice and sorghum(3-week-old plants and five plants per pot) were cagedand infestedwith three or four pairs ofadults per pot, and placed in the growttr chamber. Each pot of a host was a replication. Every 8-10 days, adults were transfen€dto pots of caged,fiesh plants while previously infestedplants were inspectedfor eggs and dead adults by dissecthg plants and searchingthrough soil with the aid of a binocular microscope. chinch bugs were allowed to lay eggs from adult emergenceto 60 days later. The averagenumber of eggs laid per female per pot was calculatedby dividing the total number of eggs by the total number of live femalesfound in a pot. The average nuorberofeggs laid per femalewas calculatedby addingthe averagenumber ofeggs laid per femaleper pot during the 60day period. The frequency distributions of eggq nymphs, and adults approximatednormality so comparisonsof developmentby stageor stadiumfor eachhost were accomplishedusing the Kolmogorov-Smimov two-tailed test (Sokal and Rotrlf 1981). The largest 'nsiped differencebetween the relative cumulativefrequencies on rice and sorghumwas comparedto a critical value @c, for c={.01). Probit analysis (sAS 1990) was used to comparehost differences in mean times and 95% confidence intervals for 50%oof the individuals to completea specific stageor stadium. A t-test or one way ANOVA (P < 0.05) (SAS 1990) was used to companecephalic capsulewidths for each instar, total nurnberof eggs laid, and preovipositiontime betweenhosts. RESTJLTSAND DISCUSSION Comparisonsof cumulative percentageof hatched eggsofirst-, second-,third- and fifth-instar nymphs,and adults showedno significant differencesin developmenton rice and sorghum (Fig. l). However, the stadium between the third- and fourth-instars was sigtificantly differ€nt for insects reared on rice and sorghun. The largest unsiped difrerenceoccurred 55 days after oviposition when 83.8 and 6l.2Yoof the total numbersof insects to reach the fourth-instar had done so on sorghum and rice, respectively. 187 Eggs o 100 €ro .t E*.0 D--o.w D--o.0?9 = 40 ho-0.0911 Dcor 0.(D3 €E E,o 23 34n Drys sftq ovipolitio! f-Ri*-----.soq$tn I | I S6md lrutar 100 $so oo Ea4E 4d'0'055 - 1" r'--o.ttt Dou -0'154 l€ 40 Dlr = 0.133 E20 o 0 34 39 44 Ddyr tfr6 ovbdilbn Fouthlnsbr 100 &80 o&* 7 F€ eo Ea' 4E AE * *. - 0.079 E€ .t'/ m=x.,T, Door - 0.093 5m tzo 0 0 44935 61 68 DrYt nrdbaili[ Dry3 dq @ipcilioo Adln r00 !80 Dil.-0.0t9 Joo D61 -022| 9lo € F20 o 61 68 Dry. .0q diPdhid FIG l. Development of chinch bugs on rice and sorghum. Data Analyzed by Kolmogorov-Smirnov two-tailed test. 188 The meantime for 50Yoof eggsto be laid or reachthe first-, second-,third-, and fifth- instars was similar for both hosts (Table l). However, the stadium betweenthe third- and fourth-instarswas 2 days longer for chinch bugs on rice than sorghum. This stadiums€emed different; the mean cumulative developmentaltime for chinch bugs on rice was not included in the 95% confidenceinterval of the meancumulative developmentaltime for chinch bugs on sorghum,- The last sampleswere inspectedfor chinch bugs at 75 days after onset of egg hatch but not all insectshad molted to the adult stage. At this time, only 45.9 and32.5Yoof fifth-instar nymphson rice and sorghunr"respectively, had molted to adults(Fig. l). TABLE l. DevelopmentalTime for Chinch Bus on Rice and Sorehum. Sorghum Stageor instar Mean 95YoC.l. Mean 95VoC.l. Egg 682 19.4 (19.1,19.7) 302 t9.4 (18.8,19.9) ltt 755 23.3 Q3.0,23.7) 535 23.2 Q2.7,23.6) 2nn 372 34.4 (34.0,34.7) 293 34.6 (34.2,35.0) 3'd 202 43.3 (42-9,43.8) 246 u.0 (43.5,44.5) 46 240 53.2 (52-7,53.7) r36 5t.2 (50.6,51.7) 56 169 60.9 (60.3,61.6) 220 60.4 (59.9,60.9) " Ihobit analysisof cumulative values; meanvalues refer to time from oviposition andg1Yo confidenceintervals (95% c.L) for 50% of individuals to reacha given stageor instar. c-ephalic . _ capsule _widths of second-, third-, and fourth-instar nymphs were significantly narower for insectsreared on rice than sorghum(Table 2). Mean differences of 14, 12, and 16 pm for cephalic capsule widths of second-, thirj-, and fourth-instar nymphs,respectively, were observed. TABLE2. Rearedon Rice and width Instar Sorghum l"' 227a 224a 20d 288b 302a 3rd 358b 370a 4th 4l8b 434a 50t 477a 48la The total numberofeggs laid for 60 daysby femalesreared on rice and sorghumwas not significantly different betweenthe hosts (Table 3). In contrast,preoviposition time was significantlylonger for femalesrearpd on rice than on sorghum(F = 15.43;df: 1,36;P < 0.05) (Table 3). Femaleson rice beganoviposition about 7 days later than did chinch bugs on sorghum. 189 TABLE 3. Ovioosition of Chinch Bue on Rice and Sorphum. AthibutenRiceonSorghum" Mean preovipositiontime (d)D l8 21.4a l8 14.3b Mean numberof eggVfemale" l8 50.5a l8 52.5a o Meansfollowed by the sameletter in a row are not sigrificantly different (P > 0.05, t-test). ' Time from adult emergenceto onsetof oviposition. " No. of eggslaid in 60 daysbeginning from adult emergence. flelayed nymphal developmentofchinch bugs rearedon corn, badey, and wheatrelative to sorghum was reported by Smith et al. (1981). Results also showed a longer stadiurt betweenthe third- and fouth-instars for chinch bugs reared on rice comparedto sorghum. Becausemost fiffh-instar nymphs had not molted to aduls, conclusionscould not be drawn regardingdifferences in developmentof fiffh-insta nymphsto adultson rice or sorghum. Similar ngrnbersof eggswere laid on rice and sorghurn,yet femaleson rice startedto lay eggs 7 days later than on sorghum. While Smith et al. (1981) found more eggs laid on sorghum than barley, wheat or com, our results revealed that the numbers of eggs each female chinch bug laid on rice or sorghum were similar. Narrower cephalic capsulesof second-,third-, and fourttr-instar nymphs rearpd on rice and a longer preovipositionperiod for adultsreared on rice suggestedthat sorghumwas a slightly better host ofihinch bug tlT was rice. Nevertheless,thc results of these experimentsindicated that rice was a suitable lost ofohinch bug. ACKNOWLEDGMENT The authorsarc grateful to Glenn Wallace, Joe Vaglic4 HanceHarper and Brandon Crausfor technical assistance;and Betsy Vaglica and Cynthia Tribble for clerical assistance; The authon also thank ttre StateofTexas (AdvancedTechnology Program), Rhone-Poulenc Ag Company,and the TexasRice ResearchFoundation for fimding the research. LITERATI.JRECITED Benton,C., and W. P. Flint. 1938. The compardive athactiv€nessof various small grainsto the chinchbug. U.S.D.A. 508: l-7. Burks,B. D. 1934.Foodplants of thechinohbug. J. Econ.Entomol.28: ll0Gll0l. crohnoln, G., A. Knutson, R Parker, G. Teetes,and B. Pendleton.1998. Managing losect and Mite Pestsof Texas Sorghuur. Publ. No. B-1220. Tex. Agri. Ext. Serv., College Station"TX. 32 pp. Dahms, R. G., R. O. Snelling, and F. A. Fenton. 1936. Effect of several varieties of sorghum and other host plants on biology ofthe chinch bug. J. Econ. Entomol- 29: ll47-1153. Flint, W. P., and W. H. Larrimer. 1926. The chinch Bug and How to Fight It. u.s.D.A' Fann.Bull. 1780:l-16. Hayes, W. P., and C. O. Jobnson. 1925. The reaction of certain grassesto chinch-bug attack.J. Agric. Res.6: 575'583. Janes,M. J., A. Iiager, and G. E. Carman. 1935. Preliminary studies on starvation and droding of-the chinch bug, Bft'ssnsleucopterus (Say). J. Econ. Entomol. 28: 638- &6. l.eonard, D. E. 1966. Biosystematicsof the LeucopterusComplex of the GenusBft'ssas (Heteroptera:Lygaeidae)' Conn. Agric. Exp. Sta.Bull.677: 147 ' t90 Painter,R. H. 1951. InsectResistance in Crop Plants.Univ. hess of Kansas,Lawtence and London.520pp. Porter, P., G.S. Cronloh, R.D. Parker, N. Troxclair, C.D. Paticlq P' Monison and T'L' Archer. 2002. Managing Insect and Mite Pests of Texas com. Publ. No. 8'1366. Tex. Coop. Ext. College Station,fi- 15 pp. SAS Institute; iS9O. SeS/jfef User's Guide, Vol. I and II, Version 6- SAS Institute' Cary,NC. smith, M. i., c. wla", and T. Mize. 1981. Chinch bug: damageand effects of host plant and photoperiod.- J. Econ. Entomol. l0:. 122'124. Snelling, n. o. ts:f. Third generation and method of migration of chinch bug in SouthwestemOklalroma J. Econ. Entomol. 2:797-803. Sokal, R R., and F. J. Rotrlf. 1981. Biometry: the Principles and Practiceof Statisticsin Biotogical Research.2ndEd. Freemanand Company'San Francisco. las V6squea- M. N., and C. Sdnchez. 191. Biologia hdbitos y huespedesde la chinchede raices Blissusleucopterus (say) (Herniptera:Lygaeidae). Rev. colomb. Entomol. 17: 8-15. Way, M. O., J. K. Olson and B. M. Drees. 2004. tnsectManagernent Altematives. 1lr Diane Bowe,nted.l 2004 Rice Production Guidelines. 8-6131. Tex. Coop. Ext. College Station,TK pp.2943. l9l vol.29 NO.3 SOUT}IWESTERNENTOMOLOGIST SEPT.2OO4 EXPRESSEDSEQUENCE TAGS AND NEW GENE CODING REGIONS FROM THE HORNFLYI'2 Felix D. Guerrero,Tim J. LysyP and Lisa Kalischuk-Tymensen3 USDA-ARS Knipling-BushlandU. S. Livestock InsectResearch Laboratory, 2700 FredericksbtugRd., Kerrville, TX,78028 ABSTRACT We have used the expressedsequence ta,g (EST) approach to isolate new gerr coding regionsfrom the hom fly, Haematobiainitots. Seventyfour unique ESTs md one completeopen reading frame were sequencedfrom genesnot previously idqtified in hom flies and recordedin the databasesof the National Center for Biotechnology Information. Fifteen ESTsare from genesinvolved in metabolism,ten ESTsare from structural proteins, and 20 ESTs are from genesinvolved in other functions, including storage,heat shock resporneand tanscription. Five of the ESTs have similarity to membersof gsnefamilies involved in resistanceto pesticides. The entire codingregion from a geneinvolved in cell cycle regulation was obtained and the mRNA e'lrcodingthe protein found to be highly expressedin eady embryonic and pupal stagesof the fly. INTRODUCTION The horn fly, Haematobia initans (L.), is m obligate blood-feedingparasite of cattle. Contolling horn flies is a major concem to cattle producers, as this pest is responsiblefor significant economiclosses to the United Statescattle industry (Kunz et al. l99l). Al0roughnumerous studies of the hom fly's biolory have beenpublished, as have oxicological studies ofthe fly's responsesto various pesticide treatnents, little is know aboutthe genomicsofthe horn fly. The horn fly possessesfive chromosomepairs (Avancini and Weinzierl 1994), and tlregenome size was estimatedat2.2X lOebp (Robertsonand Lampe 1995). The sodium charmelgw and its relationship to pyretbroid resistancehas been the focus of several studies (Guerero et al. 1997, 2002), and the sequenceof thrombostasin, an important componentin horn fly saliv4 hasbeen published (Zhang et at.2002). However, there is a tHaematobia irritans (L.) @iptera: Muscidae) \*., ar€ necessaryto report factually on available data; however, the USDA-ARS neither guaranteesnor warrants the standardofthe product, and the use of the nameby USDA-ARS implies no approval of the product to the exclusionof othersthat may also be suitable. 3lethbridgeResearch Cente, Agriculhue and Agri-Food Canada,P. O. Box 3000, 5403 l-Ave S, Lethbridge,Alberta CanadaTIJ 4Bl 193 of sequence q_ulltty data for known genesof the hom fly. Expressedsequence tags (ESTs),which are clonesselected at randomfrom cDNA libraries, rruu" pro"* very useful in the acquisition of DNA sequenceinformation for largenumben or u.tr,ropoo genes. For example, GenBankcontains 26r, 404, r02, 43r, 2g,t7g, 3,gg2, and 474 EST sequences (http://www.ncbi.nlm.nih.gov/dbEST/dbEST_summary.trrntj ror'oro sophtrtamelanogaster Meje_enAnopheles gambiae_Grles, Bombyxiori L., Amblyommau*igotu, (Fabricius), andHelicoverpa armigera (Hubner), respectively. _ This study was aimedat generatingESTs to augmentthe databaseof known horn fly.genesequences. Inlightof our laboratory's interest in tro* fly diapauseand resistance to insecticides,we used experimentalconditions which would enhancethe probability of finding ESTsinvolved in thoseprocesses. This representsthe first study of its kind in the horn fly and EST data has beendeposited in GenSankwhich can be utilized in molecular investigationsof H. irritans. The entire gene coding region to one of the ESTs was obtained and its mRNA expressionpattern in various life stagesof the hom fly was characterized. METHODS AND MATERIALS Hom flies usedto generateESTs were from a colony propagatedon a steer housed at the Lethbridge Researchcente maintained as described by Lysyk (1991) and an organophosphateand pyrethroid resistant field population fiom camp cooley Ranch, Robertsoncounty, TX, approximately 200 miles from the Kerrville laboratory. Due to interest in geneswhich might be expressedin responseto environmentalperturbations, fust- instar larvaefrom the Lethbridgecolony were exposedto l5oc for 2-5 days to induce diapauseand a control group exposedto non-diapauseinducing conditions of 25oc. The larvaewere frozen at -80oCfollowing 3, 6, or 1l daysat25oC or 5, 14,or 29 days at l5oC. These sampling points were chosen to sample both the 25oC and the l5oC larvae at equivalentdevelopmental time points basedon the resultsof Lysyk (1992) who derived the relationship betweenrate of preadult developmentand temperaturefor Alberta hom flies. Poly (A) RNA from thesesamples was isolatedusing the PolyA Pure Kit (Ambion, Inc., Austin, TX) and contaminatingDNA removedby DNAse I treatmentusing the DNA- free kit (Ambion, Inc, Austin, TX). These sampleswere utilized in differential display ReverseTranscription-Polymerase Chain Reaction(RT-PCR) experimentsperformed with considerationsfrom Simonand Oppenheimer(1996). One-hundredng of DNAse-treated poly (A) RNA was reversetranscribed with 100 units of M-MLV reversetranscriptase (Life Technologies,Inc., Gaithersbwg,MD) accordingto the supplier's instructions using the anchorprimers 5'-AAGCTTTTTTTTTTTG-3', 5'-AAGCTTTTTTTTTTTC-3', or 5'-AAGCTTTTTTTTTTTA-3' (GenHunterCorp., Nashville,TN). PCR utilized 20 pl reactionscontaining I pl of reversetranscription reactlon, I pM anchor primer, 20 pM eachof dGTP, dCTP and dTTP, 4 pM dATP, 0.08 pM c[-33p-dATp(3000 CVrnmol,l0 pCi/pl, Perkin-ElrnerLife Sciences,Boston, MA), 2.5 units AmpliTaq DNA Polymerase preparedas a l:l (vol:vol) mixturewith TaqStartAntibody (ClontechLaboratories, Palo Alto, CA), 1.5mM MgCl2,0.001% gelati4 50 mM KCl, l0 mM Tris-HCl pH 8.3 and0.5 pM of oneof the following arbitary sequencel0-mer primers,5'-AGGGA!AACAC-3', 5'- CAGGTGATGT-3', 5'-CCTCTATCGT-3', 5'-CGCTTGAGAA-3', or 5'- TGCCATTCCT-3'. Thermocycling parametersutilized an initial denaturationof 95oCfor 2 min, followed by eight cyclesof 94oCfor 30 sec, 44oCfor 2 min with 0.5oCdecrease per cycle,and 72oC for 30 sec,then 22 cyclesof 94oCfor 30 sec,40oC for 2 min, and 72oCfor 30 secand a final 72oCextension for 5 min. PCR products were fractionatedon 6% Long t94 Rangergel solution (FMC BioProducts,Rockland, ME) and analyzedby autoradiography Reproduciblegel bandscontaining DNA were cut out and DNA eluted by adding 100 pl H2O followed by incubating 10 min at 25oCthen 15 min at l00oC. Following a 2-min centifugation at 14,0009,the DNA-containing supernatantwas precipitatedwith glycogen, NlIaoAc, and ethanolaccording to standardprotocols (Ausubel etal, 1998). Four pl of the recoveredDNA was reamplified using the above protocol except that the dATP was 20 pM and eight cycles of 94oc for 30 sec,40oc for 2 min, and 72oc for 30 sec were added. PCR products were gel purified and ligated into the PCR Script Amp SK* vector and tansformed into xL-10 Gold cells (Statagene,La Jolla cA) accordingto the supplier's recommendations.ESTs which resultedfrom thesedifferential display experimentsinclude the designationDD3 or DD6 as part of their name in the Tables and in the GenBank submissions. The Camp Cooley resistant flies were enrichedfor environmentally induced gene expressionby the PCR Selectprotocol describedin Guerreroand Kunz (2000). Briefly, a subtractedcDNA library was synthesizedusing mRNA from camp cooley flies as the testersample and mRNA from Kerrville susceptiblelaboratory colony flies as a sourceof a driver subtractor. Thus, the subtractedlibrary is expectedto be enrichedfor cDNAs to genespreferentially expressedin the resistant Camp Cooley flies. ESTs which resulted from these subtraction library experimentsinclude the designationCCMP as part of their namein the Tables and in the GenBank submissions. In addition, specific genefamilies were targetedfor amplification by synthesizingdegenerate primers whose sequenceswere derivedby aligning genecoding regionsfrom various insecticideresistance-associated genes of specieswith sequencesavailable from GenBank(Table l). The Camp Cooley resistant fly cDNA was used as template for these PCRs. Touchdown degeneratepcRs were performedusing 50 pl reactionswith 1.25units Ampliraq DNA polymeraseprepared as a l:l (volvol) mixhre with raqstart Antibody (clontech Laboratories,palo Alto, cA}2 mM MgCl2,50 mM KCl, l0 mM Tris-HCl pH 8.3, 2.5 mM eachdNTp, and l0 pM each forward and reverseprimer. Thermocyclingparameters contained an initial denaturationof 96oc for 3 min, followed by 30 cyclesof 94oc for I min, 65'c for 2 min with 0.5oc decreaseper cycle, and'72ocfor 3 min, then six cycles of 94oc for I min, 50oc for 2 min, and,72"cfor 3 min and a final 72oc extensionfor 7 min. In somecases,2 pl of the pcR reaction was reamplified using identical conditions as in the first pcR. ESTs which resultedfrom thesedegenerate PCR experimentsinclude the designationRGMP as part of their namein the Tablesand in the GenBanksubmissions. Hom flies for the life stagesgene expressionexperiments with HICDRK were obtainedfrom a Kerrville laboratoryin vitro colony maintainedas describedin Guerreroet d. (1997). Variousstages of the fly's life cyclewere sampled, immediately frozen at -gOoc, and designatedas follows: Adult newly em€rge4unfed; E9: eggssampled from collection padsplaced undemeathcolony cagesfor a two-hour period then frozen; E5: eggssampled from collection padsplaced underneath colony cagesfor a two-hour period, incubatedfor 6 hours at colony rearing temperature(28oc) then frozen; 3*: third-instar larvae sampled approximatelytwo days after eggswere tansferred from collection pads to larval rearing medium; P3: l-day-old pupae sampled three days after eggs were transferred from collection pads to larval rearing medium; Pg: six-day-old pupae sampled eight days after eggswere transferred from collectionpads to larvd rearingmedium. Poly(A) RNA was purified with a singleround of selectionusing the polyAThact system 1000 @romega corp., Madison, wI) and quantified using LIV absorbance spectrophotometry. Gene expressionassays were performed with genespecific probes synthesizedfrom restrictionenzyme digested cDNA clones. T3 RNA polymeraseand the 195 U) (A i(€ -.o t x+ g =g S E -^(*)-i od h ii Y EFI od cq b - \oc\ + $ \o F € (J r) v zQ F F!) F r-F (J fr $ $ ? F P c)k z a 3 i 3 F ilF z Q,A2,?J I O L) v ESIH32S2FZ o o F l- Sl ok t Z d /, q g e4&CP{ 3?z 8X 44 P I E z F66es5? F. i 81f,E Z z v k i 3 e 7 7 F ts z FX3Pt8 u (,FO(.)HFA ol q) c) z 4Z OO()OQ .G o F G) o q) it &q o n9& c) E o eg t () q o q o( g 6 o o gg o o o * FE6 () z rq x q) Fl Ee€4€ gE () s F€>7^>H vv t96 MAXlscript In ViEo Transcription Kit (Ambion, Inc., Austin, fi) were used to g€nerate 32P-labeledRNA probes for ribonucleaseprotection assays (RPA) with the HybSpeed RPA Kit (Ambion). The RPA techniquewas chosenover a Northem blotting analysis becauseof limited samplesizes availablefor RNA isolation and RPA requires less RNA and is more sensitive than Northern blotting. Differencesin transcription levels of boti genesused as RPA probesrequired adjusfinent ofthe amountsofprobe and poly (A) RNA for eachof the geneexpression RPA o RESULTSAND DISCUSSION Our study found 75 ESTs which we were able to identiry GenBankmatches with a high degreeof confidence(Table 2). In the table, 26 ESTs are from the Camp Cooley subtractedlibrary experimentalsample and are labeled as CCMP, 17 ESTs labeled as RGMP are from the experimentsusing the genefamily-specific degenerateprimers, while the other 32 ESTs, labeledas DD3 or DD6, were derived from the diapausedifrerential display experimentswith the Lettrbridgehom flies. one-hundredand twenty other ESTs were sequenced;however, no GenBankmatch was identified and those ESTs are not listed here. As the unidentified ESTs representpotentially novel sequences,they were deposited into GenBank as horn fly EST sequencesof unknown identity (data not shown). The degenerateprimer, differential display and subtacted library approachesyielded 7, 99, and 14 ESTs,respectively, which could not be assigneda putative identity. A comparisonof the three different methodologicalsources of the ESTs shows that the degenerateprimer approachyielded the highestpercentage of sequenceswith putative identifications(RGMP ESTs,7l%), the subtractionlibrary methodologyyielded the secondhighest percentage of sequenceswith putative identifications (ccMP ESTs, 447o) while only 29yo of the sequencesfrom the differential display experiments(DD3 or DD6 ESTs) yielded putative identifications. Eighty-nine percent of the ESTs in Table 2 had e values<0.001 and thus arehighly significant matches.Our identified ESTscould be groupedas: t97 I z *\o\ood.r;ri r; n t-+c.iviXP -$Nt\o\X=-- q q q) (J l€q IH EE$EEaEExFggggH I z<< H533i;;ng ! I s I E6 l I F$ B L L\ ,.s LL\ \ l !- i{) ilo 6 IG b4 l> I I ! s$gsF$ssg$S$Fsssgrss$$ss I I {)o fr..q Iil Eor$^ I >i N er € Efi 3 €l !EcrHE E^F €Euu +g t *EEaaE ;;frfi8 EE ?IEx l.; o P.I E o E i:l b0 EEEfi€s"77$Egs$ FFFFE :E Al'6 o g€F€Eg$ig-E+Egt " 5 riaao E.=.=E3E €{€ti;;EE.F.E psEE Els A e Ei'E€ Fg gE€g 3i'!tFrts -?? ?F.Fg Pt,=,= t q .=tel tr aFX:R=KKRF888t$ o Elxa c) 0 $ilsilil$ilsns$*ssH I sXEXXXXEEEEEEEE5S$$$il$$rHHH 31fr Elc i (?)o-rq€€rfc.l oot6+oiol r\cncooool€c.| lao 6lOtoOFrsthO.l \oocno\FrNr| \oF 198 I I __l -.:..: \o \o c..l c-.1 $EEiFEEg3F$$g$HE!*g5lEg$$ $ bb$ s EE.s d-x tgXX!;Es$$€ ,*t E+ci cid-$$$$sF$sus$$$$s$$$si g g aaa;;;*$fi3:=l:l$g-E E .s "eA .s.E.EEEtrtrtrE=.E;;;;;;'; 3 E ig zzz"isg333ggggggggg3gEgf,f,ilf,ilf,EEfi$$EfrEfrHfiEfr$frfrfr8€ ? 4'|.t1111 t'li au)au)v)r .-, €cOCO€€( v H(\alN(\( e (\ o\ + O OOCIO65??UUUUUUUUU?UEEEEE$$$$$$gs$$5$$$$5$5$$$$$$$8 \o c\ e.l g3*HsHH;i sf ssrSRK$NS€H*fi$fi r99 6\o .\ci\o c.l C.l - (\| a{ :=l==F==iq*{iffixE$s €€o€€ OO+OO =$5G**il**: \O \O a{ \O \O =;\i}i9\o\o\o\o\o: C.l c"l m C{ c-l r?=RXXXXXi < 'F-FR., S**€,8 € bb EEE$ iF"F.$x.sEE$EH-sis+*$R$E€ gSE €sEs5F:it:i:i SS$ttSi€s€ss S EEB B E E $$€$ $EE$EttFE.*R'F Euouu SaoES>XXXXciN>iX?jei%clci t- F =5.E C.l 8 gE6C o € E gg EHHHHEEl EfiE ;frr ?E'o\ e.l \o c-, st t- ggggggeBEEEEEEESES5Ei*FEia (: t 6 t. s il$ssslE$As sEssssss,gEqssggEggg3eF=xsHresB:SSilX*RHHR o\ L - H H H H H H H F () X X X 8 X E - - - E -- -- E O d565U ?????< 0 () q Of-6O\6 $$ :l bi-66"-i"-r-r-F A 6r) o C) -..=-{r =EEEEbEEEF=*F***eHanIt v) ss:5iA A A N N STHHniFEEEEEEEnnAEEE'\ I r4 f \ f IHHHH V\ 200 I I I. Metabolism and enzymes (200lo),hcluding RGMP 2OSEeSand 38 and DD6-5IF which match with members of gore families with roles in insecticideresistance. Four ESTs, RGMP 20 and 40 randDD6-50C and -87E, in this group matchwith geneshaving roles in digestiveprocesses. 2. Structural components(137o), including 4 ESTs that match geneswhich aremuscle associated, RGMP 5I/52,DD3-2ZHand -76E, and DD6_31A. J. FbTd associated (3270), including ESTs from coding regions homologousto 17 ribosomalprotein genes. 4. Mitochondrial sequences(8%). 5. other Q7%o) ncruding storage proteins, including yolk protein and hexamerinhomologs, and two ESTs, RGMp ts nd 16fi1, which match geneswith a role in geneticresponses to insecticideexposure. - using the EST approach, we have successfullyidentified ."qu"n"", from several gene fanrilies which have membersthat ae invoked in pesticide iesistance, including RGMP 38, RGMP 20SEQS,DD6-51F, and RGMp 76/77 wtnchmatchwith genefamiliei in otlrerorganisms involved in resistanceto organophosphates(op), ot, pyrettroids, and !v1l9d-i9nes,respectively.- The lrimir nr-ecR "pi;*i"was especially fruitful yielding . fegenerate in ESTs which could be assignedputative identities and functions. Table I showsthat nine unique identifiable ESTs wJre isolatedusing the carboxylesterasespecific degenerateprimers. The degeneratenature of the primers rerutted in theisolation oi BSts :yding a variety of putative proteins. Theseincluded a pyruvate carborylase(RGMP 6/9)' phosphatidylinositol g), tinase (RGMP serineproteasl (RGMP 20), ihosphoserine phosphohydrolase(RGMP 2osEes), carboxylestlraselncur 3g),' chymotrypsin (RGMP 40), chorion prgFil 536 (RGMP 39), a ribonuclearprotein (nCVp 27j,'nd phosphatesotransporter (RGMP l8). The GABA r€ceptor degeneratepimer set resulted in the isolation of an EST encodinga putative GABA i"..ptoi (RGMi lalv, amougn ngithel the octopaminenor ivermectin receptor degenerateprimer set resulted in isolation of.an EST encodingthe desiredreceptor. Rattter,the octopamineand ivermectinreceptor primer sets yielded EST_sfor putative chorion protein 3rt 6cur 4l) and vacuolar sortingprotein sKDl (RGMP 63), respectively. The Drosophilamethoprene resistance qeneprimer set yielded two rmique identifiableESTs encodingputative rilosomal protein (RGMP sl4 48) andputative muscle LIM protein (RGMP si75z;. rinatty, the nematode multidmg resistancegene primer set yielded two unique identifiable esis encoding a putative member of the..wD40 repeat G protein lncrr,P g6) and succinyl-coA synthetase (RGMP 8l with sequencingperfomredwior'both the standardMr3 -40 and revenreprimers yielding two ESTsin Table I and 2). Someof the ESTs, particularly those from the ribosomaland mitochondrialgroup, matchgenes which have b31 usefirl in phyrogeneticand population studies of arthropods (Murrell et d. 2001)' Nucleotide substitutions in ribosomal or structural gene coding regions could serveas markers in these types of studies. Sodiumchannel DNA sequences from hom flies in U. s. (Gueneroet er-.2002),Mexico (Li et al. iooii*ta (Gugliglmo.ne et al. 2002) populations have bten successfully characterizedty i rcn assay developedusing U. S.flies. r"ry interesting'to "o*p.r" eit sequences to their homologues I -*"ga E from the buffalo fry, H. irritans exigua oe Iuei.yere,and rook for nucleotide differenc.esbetween these closely relateddipterirs. There are no reports that the sodium channel PCR assay has been attempted with buffalo flies. It should be mentioned tlrat althoughsome of our experimentalconditions were designedto detect gene e: 201 noted during the differential display experiments,attempts to verifr the differential gene expressionby reverseNorthern dot blot analysis and/or comparative RT-PCR revealed these differences as false positives (data not shown), a cornmon problem with the differential display technique. These ESTs can lay the foundation for a numberof future studies of the horn fly genome.The ESTsand their conespondingfull lengthcoding regionswould be valuablefor hom fly genomeand chromosomemapping studies in addition to providing probes for isolating genomic DNA fragments containing hom fly gelre promoter regions. Gene promoter sequences containing regulatory regions for expression in tissue- or developmentalstage-specific patterns would be useful for cneatingtransgenic horn fly strains expressingmarker or useful tansgene products at specific times or tissues. Additionally, the reported ESTs would be very usefirl for isolating other membersof closelyrelated genes in a genefamily of interest.For example,one could usethe DD6-51F CYP6-like cytochrome P450 EST as a probe of a cDNA library under low stringency conditionsto isolateother P450-related genes' One of the ESTs,eventually designatedHICDRK, was a matchto cyclin-dependent kinasesand related enzymeswhicir are ctiti"al to progressiveregulation of the cell cycle (Pines 1993). Li et al. (1995) found that the human GDRK homologuehals cell proliferation-in mammaliancells by stopping cell cycling in the G]{M transition' In L. insects,CDRK hasbeen cloned from D. milanogaster(Sugaya et al. 1994) andB. mori (Iwasairi et al. 1997) and studies have focusedon the role of CDRK in insect cell cycling of "nd, -o." recently, on diapause,The diapausingstage of B. raori involves a suspension of most cellular activities inciuding cell division (Kitazawaet al. 1963). Thus as a regulator into and e'dt the cell cycle, CDRK or relateiproteins could play a role in regulatingentry high during from diaiause. Additionally, as the activity of cill cycle regulators.can.\.u"ty in early periodsof rapid growttr,reiJatory regionsfrom theie g"net might be highly active for activating embryonic*a pupuf ,tage"sand wouii provide useful regulatorycomPonents Thus, the EST lifestage-specifr"tr-"g.i. activity in'transgenic hom fly experiments' the full "*oOilg tt " t om ny COnf hombloguewas targetedfor isolating and sequencing studies and length iding region using 5' RACE-PCR as a prologue to gene expression promoterisolation. bp, an The sequencefrom the full length HICDRK coding region contains 1345 at nt 53, and a open readingframe of 41i amino aciAs-inchainga methioninestart codon sequence poty (a) rriotif at the 3' end of the cDNA' A comparisono! the-aqring acid to cdc2- encodedby HICDRK with the sequencespresent in GenBankfound similarities alignmentof the relatedkinases from B. mori and6. melanogaster(Fig. l). The optimized of 395 (697o)and HICDRK aminoacid sequenceto BMCDR*. andDMCDRK shows274 (S3%) and 308 of 357 256of 357(72%) identiiies,respectively. A total of 328 of 395 ate either (86%) aligned amino acids from SIUCORK and DMCDRK' respectively' properties' identicalorconservative replacements based on amino acid side chain chemical which are The elevendomains contuinirrg the nine invariant and six nearly invariantresidues present and highly characteristicor trr. proieitr Lnase family (Hanks et al. 1988) are 210 which ae conservedin HICDRK as are tluee amino acids at positions 63, 64,,and et al' 1997)' poi"",ia sites of pfrorpt oryiation conservedin-cdc2-related kinases (Iwasaki proteins (Wierenga The domain I consensusC-X-O-X-X-C fowrd in nuoleotidebinding and Hol 1983)and the protein kinasesare also found in HICDRK' a disparity in sequencesavailable from GenBank were examinedto determineif the HICDRK and usageof nucleotide triflets encoding Tqt C{t and Gln between two speciesor if it DMCDRK was u g"n ri diff"rence in-overall.odon *"g. betweenthe (Table 3)' was specific only to proi"i* tit" CDRK with relatively low levels of expression 202 TABLE 3. Comparisonof SynonymousCodon Usagebetween Haematobia ititans and Drosophila melanosasrer. AA CODON % USACEA AA CODON %USAGEA Hib F UUU 50(51) 3e(33) Y UAU 67(66) 46 (3s) uUC 50(4e) 6r (67) UAC 33(34) s4(6s) LI.ruA 3r (22) ll (5) R CGU 4t (41) 20 (17) UUG 36(45) t7(r7) ccc 13(16) 35(35) CuU 12(10) e (e) CGA 13(14) 19(14) cuc 4 (8) l8 (16) CGG 4 (4) e (r4) CUA e (8) e (8) AGA 21(16) 6 (e) CUG e (7) 3s (44) AGG 7 (e) e(ll) M AUG 100 100 W UGG 100 100 VGuU 36(41) 2l (18) P CCU 32(33) ll (12) GUC r7 (2t) 2s (25) ccc 20(34) 32(34) GUA 22 (t8) l6 (10) ccA 3e(2s) 22(24) GUG 25(re) 3e(47) ccc 8 (8) 34(30) H CAU 6e(s7) 38(37) a cAA 74(80) 31(28) cAc (43) 3L 62(63) CAG 26 Q0) 6e(72) ACU 27(32) 11(15) A GCU 46(4s) 2t (19) ACC l8 (33) u(40) ccc 33(36) 4s (47) ACA 38(25) l5 (19) GCA 20(13) 16(16) ACG r7(10) 30(2s) GCG 2 (6) 17(18) AAU 63(60) 50(41) K AAA 64(6r) 34(27) AAC 37(40) 50(5e) AAG 36(3e) 66(73) GAU 70(72) 6e(51) E GAA 74(76) 33(30) GAC 30(28) 3l (4e) GAG 26 (24) 67(70) UCU 18(22) s (8) G GGU 37(5r) 26(21) ucc 20 (22) 2sQs) GGC 3t (25) 4e(4s) UCA r7 (t4) 12 (e) GGA 27Q2) re (27) UCG 14(12) 23 (2r) GCG 6 (2) 6 (7) AGU 20(16) t2 (t3) ACC 11(14) 17(2s) C UGU 74(60) 26(27) UGC 26 (40) 74(73) IAUU 4e(52) 33(32) AUC t8 Q7) 44(s0) AUA 22(1 aNumbers o/o indicate the use of the specific "o which can 99defor a particularamino acid. For exampl-e,in f,om ny ui.." iu.r. a 365serine (s) total of codons.of these,79 wereucu codons;thus, the z usecE of ucu is 79 + 365X 100%=22%. bcombined o/o use in the H. irritans md D. melanogaster genecoding regions for HICDRK, RAcKl, PARA,and EstEET. valuesin parentlesisi;dicate ,yrionfroou, codon usage derived froma totalof 4882and 130391 I iodons from 27 ^"a z[si itiiiiii sequencesof hornfly andD. melanogaster.respectively. 203 -.u------l- HFCDRKMCNSFLILCRMNKE|\/DP\/AP\ITF,KGYLISTKIGEPIEIRERDI/HGRCRAWEF'EKI,iIIRIGECJTYGI\ryR,ARDTRT 75 B!rDRK *SQGSEKS,ASNHEV*rrtGic*rrr\,drrFR**KprrP*KrILlKrrFrG**rrrl***'tr***tr**rotr(llf 75 tt ___l_l_ HTCDRK MWA&KKVR!,TDQEKDGLPVSGI,REITII,KKCKI{I'NWTTI,RE\/\'\'GKSI,ESICT,VMEYEEQDIASI;I,DTOIIQEEA- 150 BrcDRK GSr*r*******V****t*L***r**Q\iFldS*R*****Q*Kr*IJ**R*r**I**S*************r*SS*rT 150 mtcDlu( ***i*r*****rr******I***rr*M*rrQ*It**rtrR****t****tD*Ir*trDFrr*rr**vd***s*itT LL2 __jlt_ HFcDRK ESE'I'I(CryLQVIQGIJ(I'MHSIIYIIWPSGPMTPQV{IIXiI{I|ISPEILL zzt BICDRK **Q***Ii!****K****L**NF*V*t********L*t******r***t**$lL*ArAIlsA*iR****t*tA***** 225 IITCDRK *******T**r*KA*r*L**FlrM****t**t****i****I*V*******M*$J*!K*****M******A***** rg7 HFCDRK GSTIQTTAVDDI!{A1/GCIIGELLSHKPIJJPGNIEIAQI,EI,IIDI,IGtrPSEAIWPDYPIffPAIQNFTIJKKQPYNNI,K 300 BIICDRK Q*PRtaP*L**t*A**t**t**Al{*a****Rr**Et*r**\id*r****D****EFTSAL**L******Qerr**r* 300 Ei4CDRK rCR*H********F*****t**I6t****tts***ttDM**t*t*Attts***GFADL*i\id****sQ***ir*T 262 ------HFCDRK PKFpYIJSAAGIJRLIJNFIJFITYDPKKRATADECTIISIyFKEPPLPCDPKLMPSFPQHRMHQHNSIIOARGSSITTSM3?5 5 t) BMCDRKQR*Pllrr****************N*n*r*E*rrQ*S***tE***r****trr****r**K@TI(ATSNQINfPL*N DlrcDRK r**HMIGOS*Rll***I**I*N**T{***E***K*K**'l'fD**QAa**6{r*T**rr**}uu\DqI'qvQPPAD----- 330 TTCTNX PETPATSOITCSI,IKRKKM Jvf LN:r*****rrr *\j*I(RRIr* 402 BCDRK 348 DI4CDRK__I*_****rNVEr***Q[Of* FIG. l. Alignment of the deducedamino acid sequencefrom the HJCPRK DNA with those from the cdc2-related kinases of B. mori, BMCDRK, and D. melanogaster, DMCDRK. GenBankaccession numbers for BMCDRK and DMCDRK re D85135 (Iwasaki etal.1997),andD16402 (Sugaya et al. 1994),respectively. Asterisks (*) indfcate (-) an amino acid identity comparedtothe rucom sequence.An underscorel) or a dash indicates amino acids were omitted or a gap was inserted, respectively, to marimize alignmentto HICDRK. Locations of the I I protein kinasefamily conserveddornains ae lines above the sequence. The 9 invariant and 6 nearly invariant residues nof,"a UV "*" conservedamong protein kinasesare indicatedwith a abovethe residue' Zhartget al. (1991) have shown that in four classesof species, including primates, D" melinogaster, and the yeast Saccharomycescerevisiae Meyer ot E' C' Hansen' codon proteins' Thus' u*g" i"'ttiehly abundantproteins is different toq ttralg-ltj *u"gt Table 3 shows synonymous codon usagepooled from HICDRK and three other hom fly codingregions,".*tof*ni"nnasUeenioundtobeexpressedinadulthom fly at low levels para' (datanot shown). Thesetlree genesincluded coding regions for HictET esterase,the like sodium channeland the RACKI receptorfor activatedprotein kinase C with GenBank accessionnumbers ofAFl39082, U83872and 41304309, respectively. For comparison, codon usagein the D. melanogasterhomologues, DMCDRK, DmaE7, para' aln.dMCKI with GenBank accessionnumbers of D16402, U51050, M24285 and u96491' respectively,are includedalong with overall codonusage in all GenBankrelease 108 entries artd of completedsequences from inique protein coding regionsfrom both D. melanogaster 204 H. irritans. In the low gerr expressiongrouping which included HICDRK, the largest differencein percentageusage between hom fly andD. melanogasteroccurred in the iwo cys codons, with a difference of 4B%o(74% versus 260/ofor ucul. There were rwo codons,cuc and GCG which code for Leu and Ala, respectively, which were rarely used in horn fly but usedmore often in D. melanogaster. clJC and Gdc had % rsage valuesof 4y: ^!2o/o inhom fly and l8% and 17% n D. melanogaster.It shouldbe noted that when the codon usageof HICDRK and DMCDRK *er" "omp"r"d, the results were very similar to those obtaine{ from the comparison of the low gene expression groupin; discussedabove. The relianceupon specific codonsin the exoginoustansformin'g o\,{ which require tRNAs that occtn rarely in the host insect coddL a potential impJdiment to the.successof hom fly.tansformation experiments.If genesfrom D. melanogasrerwt,re used in transgenicexperiments with hom flies, to optimize tansgene expression,codon usagewouldneedto be carefully consideredand nucleicacid sequeicealtered to avoid the useof rare horn fly codons,particularly CUC and GCG. RPAs were used to "'ranine the expressionof HICDRK *RNA in various life stages of the hom fly' RPA techniquewas chosendue to its enhancedsensitivity compared .The to RNA Northern blotting and the limited amount of tissue availablefrom the varigus hom fly life stagcs. gel electrophoresisand ethidium bromide staining quality ]q*ut" y91qd the of the 'RNA purified from the various samples(data not shownf, HICDRK *RNA is expresscdat its highest revels in the early ;;" ;i;up"r (p3) and embryonic @e)dwetopment gtf.zal.Although nFig.2,HIciRK-autoradiographi" r"RNA appearsto be undetected pr in the adult. "4 sample, ot""o"a "*por*" showed HICDRK was expressedin those stages(data not shown). wtrei tire RpA autoradiograms were analyzed by scanningdensitometry and rhe signal correspondingto HICDRK mRNA qg3*,q "housekeeping" lgEtive ro expnessionof the enzyme GAiDH mRNA, the ratio of HICDRIVGApDH expressionwas 0.029, o.si. o1le,0.024,3.s3, na o.oru for adult, E :l' F P3 and Ps, respectively. Thus, adult and third-instar (3*) lifestageshad the lowest relative HICDRK 'RNA expression. The 6-hr embryos rnj "ira the t-aay pupal sample(Pe) had appoxirnatety G and 3-fold higher expression,respectively, than adult and third-instar larrap. Thc erly pupal (pl) and embryonic (E6) stageshad the highest by* d.H- .c?RK expressionwith values 120- and 33-iold higher,reJpecfivery, than rhe adult and third-instar samples. The early pupal and embryonicJtrg", of *r" insect'slife are characterized by high tevelsof cell divisio; and probably'requit" frgto .*pi.rrion of gene productsessential for cell cycle regulation. B. miri diapausesat the-early lib,yorri" iug" approximately one-third of embryonic development*itn tr," embryonic cells arrestedat the !rc- ]ar -fuougtt G2 stage(Nak4gFki et a. rrt;. Iwasaki "t ur. (r ssz) io*a *,ut tr,. a m.orihomologve, Bc&k'ryas e-rprgsed at significant levels in n*riy a"porit"d and 3-day- old eggsbut then dcclirpdto barelydotcctable levels by g.5 days.'ruf"t-"*p."rsion was in thar srrdy. cdrt-rtke gcnes i:j,:.Tu"d appearto aat as negativeregulaton of normal cell cyclmg around the exit to mitosis (Bunnell et al. 1990)and, as such, ii insectsmight be involved with the regulationmd eventualcessation of mltotic cycling whict occurs dgring diapause. The elevrtcd orpession of HICDRK in the early p:upJ *pr., p3, in comparisonto the other li& stagesof these flies is consistent*itn-Ai, speculation-iigt since hom flies diapausc in the pupal stageand the elevatedlevel of ntcDRK t ot to halt cycling and begin-diapause. 31u Thc GApDH 'RNA expressionresults wEre consistent Detw@nthe pupal and adult.stagcr also, though at a gr€aterlwel, in the and third instarlarval stages. 11{ % The GAPDH.I{NA expressiJnvaria:tion could have beenaue in part to variation in the overall amo'nt_o_fpoly_ (A), quantity or stored*RNA, or transcriptional activity in the different life sage RPAs.bespiti ttrosemnNe variations, the elevatedlevel of HICDRK mRNA expressionin Eg andP3in comparisonto the otherlife stasesis clear. 20s ;x* ffififfi {*tl tll* '* ** ?l tlt l* Xfm t|l*, p * t f* l1 *r}x Sr **r nril life stages' FIG. 2. RPA determinationof HICDRK g€rr orpression in various horn fly (panel p;t(D RNA was hybridized with ir virro ransaua RNA probe from HICDRK ej-'"iA'CeppH (panel B), unhybridized probe digestedwith RNAse, and hybridization pioa""t""oufyodUy a"*'t"i"i polyacryiamide gpl elecrophoresis and autoradiography' -A: rmderneath i"*ty erni.gea, ontea "a"ft"iEOt eggssanrpledfrom collection pads placed as for Eo colony cagesfoi a two hour period then frozen immediately;Ee: egp sampled pupae then incubatedfor 6 hours at28o C prior to freezing 3*: third instar larvae; P3: days after sampled3 daysafter eggswere placedon rearingmedium; P8: pupa€ sampled8 reactio:r eggewere placed on reaing medium; -: rurdlq:sted probe fro-m-c3-ntrol.RPA sample Jthout RNAse or sample ilqe.; *' contol RPA reaction with RNAse but no of the RNA. Sizeofunprotectedprobefragmentisindicatedinnucleotides(nt).Thesizes p.t""t"d HICDFk ana CApOg fragmentsare 281 and 193 nt, respectively. ACKNOWLEDGMENT we wish to thaok Mary Brumley for assistancewith nucleic acids manipulations' Grcta Buckmeierfor DNA sequencing,and Mat Waldon with hom fly rearing' 206 LITERATURE CITED Altschul, S. F., T. L. Madden,A. A. Schaffer,J.Zhang,Z. ZItar]lg,W. Miller, and D' J' Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generationof protein databasesearch programs. Nucleic Acids Res.25:3389-3402. Ausubel,F. A., R. Bient, R. E. Kingston,D. D. Moore,J. G' SeidmaqJ. A. Smith,and K' Struhl. 1998. Cunent Protocols in Molecular Biology, John Wiley & Sons, New York. Avancini,R. M. P., and R. A. Weinzierl. 1994' Karyotype of the hom fly, Haematobia irritans (L.) (Diptera,Muscidae). Cytologia 59 :269-272. Brenner, S. E. 1998. Practical databaseseuching, pp. 9-12. /z Trends Guide to Bioinformatics. Elsevier,Cambridge. Bunnell,A. A., L. S. HeathnD. E. Adams,J. M. Lahti, andV' J. Kidd. 1990. Increased expressionofa 58-kDa protein kinaseleads to changesin the cHo cell cycle. Proc. Natl. Acad.Sci. 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Thrombostasin:purification, molecurar cloning and expressionof a novel anti-thrombin protein from horn fly saliva. Insect Biochem.Mol. Biol. 32:321-330. Zhang,S., G.Zubay,andE. Goldman. 1991. Low-usagecodonsin Escherichiacoli,yeast, fruit fly andprimates. Gene 105:61-72. 208 vol.29 NO.3 SOUTTIWESTERNENTOMOLOGIST SEPT.2OO4 rsrgO tRgttUENTS FOR CONTROLOF INSECTPESTS OF SORGHUMAND THEIR EFFECTON YIELD G. Wilde,K. Roozeboomt,M. Claassent,K, Janssent,M. wiftl, P' Evansl, T. Harveyl,and J. Long2. Deparfinentof Entomology,Kansas state university, Manhattan, KS 66506-4004 ABSTRACT Tbree seedfieatrnent insecticideswere evaluatedfor their effect on greenbugs, Schizaphisgraminum (Rondani),and clinch bugs, !ft'ssns leucopterus(Say), during a three-yearpi.ioO. Ctothianidin(Poncho), imidachloprid(Gauchoo), and thiomethoxam (Cruiser@)were equally effectivefor controllingboth kinds of pests.Differences were detected when various rates of the insecticides were used. Two insecticides (imidachloprid and thiomethoxam) were evaluated for their effect on yields of two sorghumhybrids in the presenceor absenceof insectpests at six locationsin Kansas during a three-yearperiod (2000-2002).Significant differencesin yield betweentreated and nonfieatedsorghum occurred at tbreeof five locationsin 2000, one of six locationsin 2001, and one of six locations in 2002. Sorghumyield in responseto insecticidal treafinent differed most often at Hesston (two of three years), a location frequently infestedby chinch bugs. Damagingnumbers of chinch bugswere detectedthere in one of the two yearswhen sorghumyields differedas a resultof insecticidaltreatments. These results were similar to those of a previous study in which insecticide-treatedsorghum yielded significantly more than nontreatedsorghum in tbe presenceof an insectpest. No significantdifference in yield betweenplots treated with Cruiser'or Gauchb*was found, ex-ceptat one site in one year, when plots treatedwith Cruiser@yielded significantly more. INTRODUCTION The chinchbug,8/isszs leucopterous leucopterous (Say), has been a major insect pest of sorghum, Sorghum bicolor (L.) Moench, in Texas, easternKansas, and southeastemNebraska for many years.Chinch bugs causedannual lossesofas much as $11.3and l0 million in 1990and 1991,respectively, in Nebraska(Spike et al' 1991). Chinch bugs havebeen managedby a combinationof cultural practices(e.g., planting sorghumaway from wheat) and insecticides,primarily imidacloprid treatedseeds (Wilde 1997). The greenbug,Schizaphis graminun (Rondani),has been a major pest of sorghum throughout the Great Plains region of the United Statessince 1968 when it was first observedto damageiorghum in Texas, Oklahoma,and Kansas(Harvey and Hackerott 1969).Since that time, it hasbeen managed by a combinationof insecticideapplications ' DeparEnent ofAgronomy, Kansas State University 2 SoutheastAgdcultural ResearchCenter 209 and resistantplants. However, the discoveryof new biotypes(Harvey ' et ar.- l99l) and resistanceto insecticides(Archer 1994)necessitated new uiprou.t"r. Imidacloprid, "reduced . . thiamethoxam,and clothianadin are so^me of the new risk,, insecticides that will increasein importanceas altemativesto orgu;pnorphates and carbamates. Becauseonly the seedis treated,less active ingredientis apptiedper acre, and risks associatedwith sprayapplications, such as human-exposurein'tle mixing and applicationprocess andpesticide drift, areminimized. The developmentof new systemic insecticideshas generatedinterest in the use of seedtreatments ibr control of sorghum insectpests. Cruiser@is a trademarkof thiamethoxam,a second-generationneonicotinoid insecticidedeveloped by syngentacrop protectionkrc. (Greeisboro,NC). It provides excellentcontrol of a wide varietyof commerciallyimportant insect pests on a varietyof crops including barley (Hordeum vulgare sussp. vulgare), cotton (Gossypiumspp.), iom (Zea mays), wheat (Triticum aestivum)and canola (Brassica napus), utro *o.ts tttottgt contact,stomach and systemic^activity (Wilde et al. 2001). , Imidacloprid(Gaucho) is commonlyused as a systemicseed treatment ro protect seedsand seedlings against injury by earlyseason insects (Pike et al. I 993, Sloderbecket al._1996'wilde 1997,Tharp et al. 2000).Marketed by GustafsonInc. @lano,TX), it is effectivein controllingmany insects, including aphids (Aphiidae), thrips (Thysanoptera), and wireworms(Elateridae) and is commonlyused on severalcrops, including cotton, wheat,barley, com, canola,and sugar beets (Beta vulgari). Ponchcj-(clothianidin)seed-applied insecticide also is producedby Gustafson.It is a second-generationchloronicotinyl insecticide (CNI) which has excellentsystemic activity and doesnot adverselyaffect seedgermination or the crop. Insectstreated with clothianidindevelop no cross-resistanceto insecticidesofother chemicalclasses. Limited informationis availableon the yield of sorghumgrown fr_omseed treated with insecticidewhen insect pests are scarce. Previous work with Gaucho@on sorghumat Hesston,KS, showed that yields differed among-445.i sorghum hybrids. Sorghumfrom Gaucho@treated seed ptantla in April yielded and A1O.Stcg/ha'more than nonheatedsorghum in 1996 and 1997,respectivelS and 763.2 and 890.4 kg/ha more when the sorghum was planted in May. A few chinch bugs were present in these experiments.However, in similar testsat four other locationsin the state,little or no impacton sorghumyields was foundwhen insects were scarce (Wilde et al. 1999). Seedtreatments would be particularlyuseful when one or more pests are a chronic problem.The objectivesof this study were to evaluateseveral seed-treatment insecticidescurrently registered or being testedon sorghumfor their effectson chinch bugs and greenbugsand to determinethe effectsof the insecticideson felds of two sorghumhybrids grown in the Midwest. MATERTALSAND METHODS Eighteentests were conductedduring 2000-2002at six locationsassociated with KansasState University. The locationswere the EastCentral Experiment Field, Ottawa; the Agronomy North Farm, Manhaftan;the Harvey County Experiment Field, Hesston; the SouthwestResearch-Extension Center, Garden City; the Northwest Research- ExtensionCenter, Colby and the SoutheastAgricultural Research Center, Parsons. Each locationhad different soil typesand agronomic practices. Treated and nonheated seedsof two sorghum hybrids (NC+ 271 and NK KS 560Y) were obtainedfrom the respectivecompanies. Each hybrid was treatedwith imidacloprid(l.l ml/kg) and thiomethoxam(0.7 ml/kg). Two-row plots, 6.7m long and 0.76m apart,were arrangedin a randomizedcomplete block replicatedeight times. 210 Reducedtillage in combinationwith herbicideswas used to control weeds' Normal fertilization piactices were used. Soil moisfure at planting was adequatefor seed germination and plant emergence.Planting date and agronomicinformation are given in Tablel. TABLE l. Agronomic Informationfor Evaluationof InsecticidalSeed Treatments on Sorehum- in Kansas,2000'2002' Locatron Ynl 2ooo Manhattan l0 May 15 Sep Soybeans Ottawa 12 May 13 Sep Soybeans Parsons 19 May 13 Sep Soybeans Hesston 29 Apr I Sep Wheat Colby lost due to poor standsand eaiy fteeze GardenCity 15 May 14 Oct Fallow 2001 Manhattan 14 May 28 Sep Soybeans Ottawa 8 May I Oct Soybeans Parsons 14 May 13 Sep Soybeans Hesston 2May 13 Sep Soybeans Colby 24May 5 Sep Com GardenCity 16 May 30 Oct Fallow 2002 Manhattan 2l May 30 Sep Soybeans Ottawa 23 May 18 Sep Soybeans Parsons 24 Apr 6 Sep Soybeans Hesston 7 May 17Oct Soybeans Colby 29 May 6 Sep Sorghum GardenCity 23 May 20 Nov Fallow Various growth parameterswere measuredto evaluatesorghum response to the insecticidal treafinents.Plots were examinedat two-week intervals for the presenceof insects.When infestationsoccurred, the numberof insectsper plant or a damagerating was determinedby examiningfour plants in each plot. Plots were harvestedwith a modifiedGleaner combine (AGCO Corp,,Duluth, GA), andyields andtest weights were determinedwith an on-boarddata collection system (Harvest Master, Logan, UT). The data were subjectedto analysisof varianceusing PROC GLM (SAS Institute 1998). Means were separatedutilizing Fisher'sprotected LSD with a 5%olevel of sigrificance. The effectsofvarious insecticidalseed treatments were assessed in a field heavily infestedwith chinchbugs on the AgronomyNorth Farm,Manhattan, KS. A v-belt seeder was usedon ll June2001 to plant sorghum(various hybrids) in one-rowplots, 4'6m long, in a randomizedcomplete block designwith four replications.On I I July, the numborofchinch bugsper plantwas counted on oneplant in eachreplication, and a scale of 0-10,where 0 = no damageand l0 = all plantsdead, was usedto ratethe whole plot. Yield wasdetermined by usinga smallplot harvesteron l0 September. 2tl In2002, chinchbug conhol on seedlingsorghum was evaluatedat two locations. At upland, KS,20 one-rowplots 9.lm long wereplanted on 7 June2002 in a randomized completeblock designwith four replications,for a total of 80 plots, The 0-10 damage rating scale was used on I July 2002 to evaluatethe effecti of the insecticidesin conholling chinchbugs on the sorghum.Plots were harvested and yield was determined on 25 september.In anothertest, seed treatments Gaucho@ and ponctro@were compared for chinchbug controlin plots plantedat Galva,KS, on I I June2002. The 0-10 damage rating scalewas usedon 5 July to evaluatethe effectsofthe insecticides.No yield data were taken becauseplgts were destroyedon l0 July. ln other plots plantedin another field at Galva,Gaucho'and Cruiser'were comparedusing the sameprotocol. Various seed treatmentsand severalgranular and liquid insecticidesapplied infurrow were evaluatedin a field infestedwith greenbugsat the Agricultural Research Center,Hays, KS. Sorghumwas plantedon 22 May 2001 in one-rowplots, 4.6m long, replicatedfour times in a randomizedcomplete block design.The numberof greenbugs per plant on five plantsper replicafionwas countedon 2 and 6 Juneand on four plants per replicationon 24 July. Yield was not determinedbecause of damageby birds and raccoons.ln 2002, control of greenbugsand corn leaf aphids,Rhopalosiphum maidis (Fitch), was evaluatedat Hays in plots planted on 16 May 2002. The number of greenbugsper plant on four plantsper replicationwas countedon 6 Junewhen plants were in the four-leaf stage.Control of com leaf aphidswas evaluatedon 17 July by countingthe numberof plantsheavily infested (>200 aphids per plant) in the plots when plants were in the late-whorl stage.Yield was not determinedbecause of damageby birds. All datawere analyzed by usinganalysis of variance.Mean separationwas based on a Fisher'sprotected LSD testat a probabilityof 0.05(SAS Institute 1988). RESULTS Results of factorial analysis indicated significant seed treatment-by-hybrid interactions for various parametersin each year. Therefore, data are presented for individualseed treatment-hybrid combinations as well as seedtreatment means for each year. Significanteffects of the insecticideson yield were detectedat threeof the five locationsduring 2002 (Table2). Data were not takenat Colby becausesevere drought preventeduniform standestablishment. Yield was greateras a result of seedtreatment particularly at Hesston, where the sorghum was infested by chinch bugs. Both insecticidessignificantly reduced abundance of chinch bugs, resulting in significantly greaterplant vigor (Table 3). There was no significant differencein yield between sorghumtreated with the two insecticides.At Hesston,both insecticidesresulted in greateryields of NC+ 271 andNK KS 560Y by an averageof 966.7and 413.1 kg/ha, respectively.The greateryeld at the Parsonssite was almost as much as at Hesston. Similar effectson othertraits measuredwere detectedbut differenceswere usuallvless. At Hesston,for example,both sorghumhybrids treatedwith Cruiser@and Gaucho@ reachedthe half-bloomstage in slightly fewerdays then did nontreatedsorghum, and the weight of NC+ 271 per bushelwas slightly more when the sorghumseeds were treated with either insecticide,but NK KS 560Y showedno analogousresponse. Insecticidal treatmentof the seedresulted in a sigrificantdifference in standestablishment at eachof the locations. In 2001,significant effects ofthe insecticideson yield were detectedin only one (Colby) of six locations(Table Q. While no insectswere observedat this location, 212 sorghumseed treated with Cruiser@and Gaucho@resulted in greateryield of NC+ 271 and NK KS 560Y by an averageof 438.8 and 858.6kglha, respectively.NK KS 560Y treatedwith Cruiser@yrelded significantly more than the same hybrid treatedwith Gaucho@,but there was no significant difference in yield of NC+ 271 when either of the two insecticideswas used.The lack of insectsat somelocations and severedrought at TABLE 2. SorghumSeed Insecticide Comparisons,2000. -:- Colby City Treatnent' Hybrid Manhattan Ottrawa Parsons Hesston irrigated irrigated Stand(%) Untreated NC+271 113.7 121.4 87.3 105.3 100.7 Gaucho NC+271 115.4 131.4 85.0 105.5 93.8 Cruiser NC+271 116.4 r3r.2 93.8 110.0 106.8 Untreated NKKS 560Y 109.8 l3l.9 84.8 110.4 92.6 Gaucho NK KS 56OY92.8 108,3 82.3 9 3-4 85.9 Cruiser NKKS 560Y 112.2 134.8 88.2 tt7.l 104.0 LSD(0.05) 3.6 9.0 NS 6.4 -- 6.6 Trt. + Hybrid interaction Sig Sie NS Sig -- NS Untreated ttt.7 126.7 86.1 r07.9 96.7 Gaucho 104.1 119.9 83.6 99.4 89.9 Cruiser .l14.3 133.0 91.0 113.5 105.4 TreatmentLSD J.U 7.6 5.3 5.3 ).o Daysto halfbloom Untreated NC+271 67.4 67.0 60.8 77.8 7t.l Gaucho NC+271 67.0 67.0 61.0 76.3 70-8 Cruiser NC+271 67.0 67.0 60.6 76.6 71.1 Untreated NK KS 560Y 63.8 64.1 59.5 72.5 64.9 Gaucho NKKS 56OY 64.4 64.t 58.6 72.1 65.4 Cruiser NK KS 560Y 63.5 63.5 58.9 72.0 64.9 LsD(0.05) 0.4 0.3 0.4 0.3 0.9 Trt. * Hybrid interaction sig Sig Sig Sig NS Untreated 65.6 65.6 60.1 75.r 68.0 Gaucho 65.7 65.6 59.8 74.2 68.1 Cruiser 65.3 65.3 59.8 IC.5 68.0 Treahnent LSD NS 0.2 NS 0.3 NS Yield(kglha) Untreated NC+271 8815.6 8872.8 579t.4 6257.6 5954.2 Gaucho NC+271 8744.4 9066.8 6438.9 72t8.6 6132.3 Cruiser NC+271 8656.0 9077.6 6283.7 7226.2 6402.6 Untreated NK KS 560Y 8406.0 902r.7 6410.9 6476.4 4527.0 Gaucho NK KS 560Y 8850.6 8946.0 6736.5 6822,4 4680.3 Cruiser NK KS 560Y 8733.6 9595.3 7045.6 6956.6 4749.0 LSD(0.05) NS 364.4 384.1 199.7 416.6 Trt. i Hybrid interaction NS NS NS Sig NS Untreated E6r0.E 8947.2 6101.1 6367.0 5240.6 Gaucho 8694.8 9006.4 6587.7 7020.2 5406.0 Cruiser 8797.8 9336.5 6664.6 7091.4 557s.8 Treatrnent LSD NS 304.6 32s.6 166.6 NS 213 TABLE 2. Continued Garden Colby City Treatmentr Manhattan Ottawa Parsons Hesston irrisated inisated Testweight (lb/bu) Untreated NC+271 60.42 60.86 59.73 60.70 60.45 Gaucho NC+271 60.15 50.91 59.8s 6t.13 60.49 Cruiser NC+271 60.01 61.11 59.85 6t.09 60.45 Untreated NK KS 56OY 60.30 6r.54 60.29 61.s6 60.50 Gaucho NK KS 56OY 60.03 61,59 60.s0 61.58 60.70 Cruiser NK KS 56OY 60.00 61.44 60.40 61.35 60.75 LSD(0.05) NS 0.36 0.19 0.2r 0.l9 Trt. t Hybrid interaction NS NS NS Sig NS Untreated 60.03 61.2 60.01 61.13 60.48 Gaucho 60.09 61.25 60.18 61.35 60.59 Cruiser 60 6r.27 60.13 61.22 60.60 Treatrneflt LSD NS NS NS NS NS "Gaucho@ appliedat 1.1 rrl of Gaucho@480&9. Cruiser@applied at 0.7 rnl of Cruisero5 FS/kg. TABLE 3. Effect of Ssed-treatmentInsecticides on C@ * Untreated NC + 271 8.1 3.1 Cruiser NC + 271 3.2 1.2 Gaucho NC + 271 4.4 t.2 Untreated NK KS 560y 6.8 3.2 Cruiser NK KS 560v 4.4 l.l Gaucho NK KS 560y 2.3 1? LSD1.5 1.9 0.26 uGaucho@ applied at 1.1 ml of Gaucho@480&9. Cruiseroapplied at 0.7 ml of Cruiser@5 FS/kg. oAvg. no. on 4 plantVreplicate. " Vigor scoreon 30 May, I : good,5 = poor. othersmay have accountedfor lack of any insecticidaltreatment effects on yield. For example,yields of sorghumat Hesstonand Ottawain 2001 were abouthalf of thosein 2000.Nevertheless, stand establishment of treatedsorghum was sigrificantly betterthan nontreatedsorghum at four ofthe six locations. 214 TABLE 4. SorehumSeed Insecticide Comparisons,200l. Garden ColbY CitY Treatrnenf Hybrid Manhattan Ottawa Parsons Hesston Irrigated lrigated Stand(7o) Untreated NC+271 140.9 141.5 59.1 102.8 98.3 70.1 Gaucho NC+271 t36.7 t40.4 69.8 103.3 93.1 76.8 Cruiser NC+271 138.8 142.6 65.2 99.6 92.5 76.0 Untreated NK KS 550Y 133.8 133.2 66.7 39.9 96.1 62.3 Gaucho NK KS 560Y t19.7 125.5 71.4 56.8 80.1 35.3 Cruiser NK KS 560Y 126.1 132.3 68.3 52.4 94.5 62.2 LSD(0.05) 4.8 7.t NS 6.8 8.5 8.0 Treatment*Hybrid (Ff) 0.0 Ns Ns 0.0 0.0 0.0 Untreated t31.3 137.4 62.9 7t.4 97.2 66.2 Gaucho 128.2 133.0 70.6 80.1 86.6 56.0 Cruiser t32.4 t37.5 66.8 76.0 93.5 69.1 TreatmentLSD (0.05) 5.4 NS NS 4.8 6.0 5.6 Daysto halfbloom Untreated NC+271 72.0 68.5 70.r 77.5 81.4 Gaucho NC+271 72.2 67.8 69.9 76.4 76.9 79.9 Cruiser NC+271 71.8 68.4 69.9 76.9 77.4 80.0 Untreated NK KS 56OY 67.7 64.s 68.4 76.8 71.0 76.4 Gaucho NK KS 56OY 67.3 64.5 68.6 75.0 70.4 76.8 Cruiser NK KS 56OY 67.8 64.0 68.4 75.8 70.3 76.4 LSD(0.05) 0.6 0.8 0.9 1.3 0.6 r.2 TreatmenttHybrid (F.F) NS NS NS NS NS NS Untreated 69.8' 66.5 69.3 77.0 74.3 78.9 Gaucho 69.8 66.t 69.3 75.7 73.6 78.3 Cruiser 69.8 66.2 69.1 76.3 73.8 78.2 TreaanentLSD (0.05) NS NS NS 0.9 0,4 NS Yield (kglha) Unteated NC+271 7322.9. 44'48.8 6068.1 3410.9 8735.5 6755.6 Gaucho NC+271 7446.3 4805.6 7234.s 3595.9 90rr.5 7343.3 Cruiser NC+271 683s.7 4798.6 6768.3 3436.9 9342.2 7424.7 Untreat€d NK KS 56OY 6513.9 3842.7 66602 2706.2 8282.0 7663.2 Gaucho NK KS 56OY 6937.s 4383.3 6366.4 3107.5 89r8.0 6830.6 Cruiser NK KS 56OY 7001.1 4696.9 5835.9 3038.8 9366.4 7013.2 LSD(0.05) 467.5 NS NS 333.9 388.0 NS Treatnent+hybrid (FF) 1.3 NS 2.5 NS NS NS Unheated 6918.4 414s.8 6364.1 3058.5 8s08.7 7209.4 Gaucho 7lgt.g' 4594.5 6800.4 3351.7 8964.7 7086.9 Cruiser 6918.4 4747.7 6302.1 3237.9 9354.3 72t8.9 Treatnent LSD (0.05) N8 NS NS NS 274.t NS 2'15 TABLE 4. Continued Garden Colby City Treatment" Hybrid Manhattan Otlawa Par.sons Hesston Irrigated lrrigated Test weight (lb/bu) Untreated NC+271 52.7 55.2 57.9 58.1 59.2 60.9 Gaucho NC+271 51.9 54.7 58.9 57.5 59.4 60.8 Cruiser NC+271 52.5 ss.4 58.0 57.7 59.2 60.9 Unteated NK KS 560Y 50.0 52.6 57.5 57.5 59.5 60.4 Gaucho NK KS 56OY 5I.5 53.4 57.7 58.0 s9.2 60.5 Cruiser NK KS 560Y 52.3 53.4 58.2 58.0 59.4 60.5 LSD(0.05) NS r.2 NS NS NS 0.2 Trcatment*hybrid (FF) NS NS NS O.O NS NS Untreated 51.3 53.9 57.7 57.8 59.3 60.6 Gaucho 51.7 54.1 58.3 57.8 59.3 60.7 Cruiser 52.4 544 58'l 57.9 59.3 60.7 TreatmentLSD (0.05) NS NS NS NS NS NS "Gaucho@ appliedat 1.1 ml of Gaucho@480/kg. Cruiser@applied at 0,7 ml of Cruiser@5 FS/kg. Significant insecticidal treatment effects on yield were noted at only one (Hesston)-ofthe six locationsin 2002(Table 5). while chinchbugs were observed in the area.thev were not abundant.At this location,NC+ 271 and NK KS 560Y sorghum treatedwith Gaucho@and Cruiser@yielded 578.'7and 591'4 kglha, respectively,nrore than nontreatedsorg[um. Sorghumteated with Cruiser@yielded an averageof 699.6 kg/ha more than non treatedsorghum, which was siglificantly q:eater-thanthe 477.0 kilha gainobtainedfrom sorghuritreaied with [email protected] and Cruiser@had no rignifti-t effect on the othersorghum param€ters measured at most locations,but there was a significantinsecticidal treatrnent effect on standestablishment at five of the six locations. Lack of insects and the severe drought might have affected the ability of hybridsto respondto insecticidaltreatment' : All insecticidaltreatments in 2001 significantly(df = 19, F : 19'70,P 0'001) roducedchinch bug abundancerelative to the four nontreatedchecks when plantswere infestedas seedlings(Table 6). None ofthe insecticidaltreatments differed significantly in their effect on abundanceof chinch bugs. Similar results were noted for damage = 7'49, P = ratings,although DK 36 and P8500hybrids were significantly(df l?: ! ,= 0.00i) moredamaged than the othertwo (NC+ 271 andNK KS 560Y).While significant = differencesin damageoccuned yield datawere variable (df.= 19,F = 3.46,P 0.0001). This is not uncommonin experimentswith chinchbugs where abundance of chinchbugs is variableand resultingdimage may be severein one plot and much less in anottrer (Wilde1997). 2r6 TABLE 5. SorghumSeed Insecticide Comparisons, 2002. Garden Colby Citi Treatnent' Hybrid Manhattan O$awa Parsons Hesston Irrigated lffigated Average Stand(%) Untreated NC+271 99.9 84.9 71.7 58.8 73.5 46.5 72.6 Gaucho NC+271 t0/'.9 96.0 75.4 74.3 78.1 s4.0 80.4 Cruiser NC+271 104.4 99.1 84.7 79.6 79.0 46.7 82.2 Unteated NKKS560Y 110.5 79.4 64.8 53.8 75.0 55.1 73.1 Gaucho NK KS 560Y 86.3 87.3 6r.6 69.2 55.1 44.2 67.3 Cruiser NK KS 560Y 108.0 91.4 74.8 78.0 74.5 49.4 79.3 AVEP.AGBS102.3 89.7 72.2 68.9 72.5 49.3 7s.8 cY(yo 7.4 9.7 12.0 9.0 8.r 12.8 9.6 LSD(0.05) 7.6 8.8 8.8 6.3 6.0 6.4 2.9 Treatnent*Hybrid (F^F) 0.0 NS NS NS 0.0 0.0 0.0 Untreated 10s.2 82.2 68.2 56.3 t1.5 50.8 72.8 Gaucho 95.6 91.? 68.5 71.7 66.6 49.1 73.9 Cruiser 106.2 95.3 79.8 78.8 IO,I 48.1 80.8 Treatnent LSD (0.0s) ).4 6.2 A1 4.4 NS 2.1 Daysto halfbloom unreated Nc+271 63.8 63.3 83.9 75.3 37.6 67.6 6s.2 Gaucho NC+271 63.1 62.9 83.3 75.3 37.1 67.6 64.9 Cruiser NC+271 63.0 62.9 83.0 74.9 37.6 67.5 64.8 Unheated NK KS 560Y 59.9 60.1 92.4 73.4 29.6 64.1 61.6 Gaucho NK KS 560y i9.9 60.0 82.4 73.0 3l.l 64.0 61.7 Cruiser NK KS 560Y 60.1 60.0 92.1 73.0 29.9 64.1 61.6 AVERAGES61.6 61.5 82.8 74.1 33.8 65.8 63.3 cvva 1.0 0.7 0.9 0.6 3.1 0.7 1.0 LSD(o.05) 0.6 0.4 0.8 0.5 0.5 0.3 Treatrnent*Hybrid(FF) 0.1 NS NS NS 0.0 NS O.O Untreated 61.8 61.7 83.1 74.3 33.6 65.9 63.4 Gaucho 61.5 6r.4 82.8 74.1 51, I 65.8 61 1 Cruiser 61.6 6t.4 82.6 73.9 33.8 65.8 63.2 Treatment LSD (0.0s) NS ws Ns NS NS NS 0.2 217 TABLE 5. Continued Garden Colby City Treatment" Hybrid Manhattan Ottawa Parsons Hesston lrrigated Irrigated Average Yield (kglha) Untreated NC+271 5136.3 4123.8 4198.2 2669.3 11779.4 8706.8 6102.3 Gaucho NC+271 5523.7 4260.6 3861.8 3128.5 11980.38670.6 6237.6 Cruiser NC+271 5402.8 4320.3 4544.2 3368.3 11907.88868.4 6402.0 Untreated NKKS 560Y 5334.1 362'7.7 3686.3 2669.9 t0544.9 8205.7 5678.1 Gaucho NK KS s60Y 5423.8 3710.4 3924.8 3161.6 10690.58399.7 5885.1 Cluiser NK KS 560Y 5478.5 3820.5 4057.7 3367.0 10542.38551.0 5969.5 AVERAGES5383.1 3976.9 4045.6 3060.4 1r240:l 8566.9 6045.6 cv(%) 616.9 503.1 978.8 505.6 334.5 437.6 533.0 LSD(0.05) NS 319.3 NS 246.8 600.4 598.5 203.s Treatmeut*Hybrid(FF) NS NS NS NS NS NS NS Llntreated 5235.6 3875.8 1942.6 2669.9 11162.4 84s6.3 5890.0 Gaucho 5474.1 39E5.83893.6 3145.0 11335.48535.1 6061'l Crurser 5441.0 4070.4 4301.3 3367.6 11225.48710.0 6lEs'7 'I'reahnent LSD (0.05) NS NS NS t74.9 NS NS 143.7 Test weight (lb/bu) Unreated NC+271 58.1 59.8 58.3 60.0 59.2 60.1 59.2 Gaucho NC+271 58.3 59.5 59.0 60.0 59.0 60.1 59.3 Cruiser NC+271 58.2 59.1 58.3 59.5 59.2 60.0 59.1 Untreated NK KS 56OY 57.3 58.2 59.2 59.2 61.2 60.1 59.2 Gaucho NK KS s60Y s6.9 58.5 58.8 59.6 61.1 60.1 59.2 Cruiser NK KS 56OY 57.9 58.3 59.0 59.7 60.7 60.2 59.3 AVERAGES 57.8 58.9 58.8 59.7 60.1 60.1 s9.2 cv(%) 1.0 1.2 2.8 r.2 0.8 0.2 r.4 LSD(0.O5)0.6 0.7 NS 0.7 0.s 0.1 NS NS Treatrnent+Hybrid(Ff) 0.0 NS NS NS NS NS 59.2 Untreated 57.7 s9.0 58.8 59.6 60.2 60.1 59.2 Gaucho f /.o 59.0 58.9 59.8 60.1 60.1 60.1 59.2 Ctuiser 58.l 58.7 58.6 59.6 60.0 u caucho@applied at 1.1 ml ofGauchoo 480/kg. Cruiser@applied at 0.7 ml ofgruiser@5 FS/k'' 218 TABLE 6. Chinch Bug Contol on Sorghumwith Treated Seed and Soil-Applied Insecticides,Manhattan, KS,2001. . Treatnent Hybrid _&rrm" Rate CB/plant DRb'" kg/hac Untreated 271 20.0a 3.8b 4432.9bcde Gaucho480 271 ST l.l ml/kg 0.5b 0.8c 5946.6abc Cruiser 271 ST 2009/100 kg 0.5b 0.3c 6436.3abc Untreated 560y 20.3a 3.5b 5495.0bcd Gaucho480 560y ST l.l ml/kg 4.0b 0.0c 6391.8abc Cruiser 560y ST 2009/100 kg 0.0b 0.3c 4738.2bcde Furadan 271 4F 02m1/m 2.0b 0.3c 8217.la Temik 271 l5G 0.9kg/ha t.3b 0.5c 6843.4ab Unreated DK-36 16.5a 6.8a t354.7f Gaucho480 DK-36 ST l.l ml/kg 2.3b l.3c 4242.lcde L0263-Ar DK-36 ST 0.7 mlkg l.5b 0.8c 4477.4bcde Clothianidin600 DK-36 ST 2000ppm 0.0b 0.0c 4210.3cde Clothianidin600 DK-36 ST 2500ppm 0.0b 0.3c 5l l9.8bcde Clothianidin600 DK-36 ST 0.13ml/kg 0.3b 0.3c 5673.1abcd Gaucho480 DK-36 ST 0.2m1/kg 0.3b 0.3c 4560.lbcde Temik DK-56 o.ekg/ha l.0b l.0c 2817.5ef Untreated P8500 ]:o 17.5a 6.5a 1138.4f Gaucho480 P8500 ST 250g/100kg l.3b 0.3c 3383.5def Cruiser5FS P8500 ST 200g/l00kg l.8b 0.5c 475Q.9bcde Temik P8500 l5c 0.9ke/ha 1.8b 0.0c 4509.2bcde t ST= Seedtreafinent. bDR= Damagerating scale, 0-10, 0 = no damage,l0 = severedamage. "means with sameletter are not significantlydifferent a p < 0.05. Chinch bugs severely damagedsorghum plots at Upland in 2002, and alt insecticidaltreatments resulted in significantly(df = 19,F:9.79,p = 0.001)Iess damage by chinch bugs.Damage by chinch bugs did not differ betweensorghum plots treated with infurrow granular(Temik) or liquid (Furadan)insecticide br seedtreatments of imidacloprid(Gaucho@), thiomethoxam (cruisere), and clothianidin (ponchoo) (Table 7). Again, yield was variablebecause of variationin abundanceof chinch bugs,but most treatedsorghum yielded considerably more than the nontreatedplots (df = 19,F = 4.7,p = 0.0001). 219 TABLE 7. ChinchBug Controlwith TreatedSeed and Soil-Applied Insecticides, Upland, 2002. t Treatment Hvbrid Form Place Rate DR Unteated K S 560y 9.5a 394.3c Gaucho480 K S 560y 4FL ST l.l tul&c 3.0b 3886.0ab Poncho K S 560y ST 2000ppm a r.3b 6385.4a Temik K S 560y 15G IFG 0.9kglha 0.0b 6162.8a Counter K S 560y 15G IFG 0.9kglha 4.0b 4210.3ab Furadan K S 560y 4F IFL 0.2m[/m 0.3b 5819.4a Unfeated DK 54 9.5a ll57.7bc Gaucho480 DK 54 FS ST 250gal1000 kg seed 0.5b 4941.7a Cruiser DK 54 5FS ST 200 gal1000kg seed l.8b 4541.0ab Counter DK 54 20 CR IFG 5.6gal100 m 2.0b 4979.9a Furadan DK 54 4F IFL Q.2mUm 0.3b 6Q99.2a Untreated NC +271 9.3a 1004.9bc Gaucho NC +271 4FL ST 1.1mVkg 2.3b 4280.3ab Cruiser NC +271 5FS ST 200gal1000 kg seed 0.8b 4197.6ab Temik NC +271 15G IFG 0.9kglha 3.0b 4534.7ab Counter NC +271 15G IFG 0.9 kg/ha 3.0b 3428.Qab Furadan NC +271 4F IFL 0.2 ml/m 0.0b 4852.7a "ST = Seedtreatment, IFG = infunow granular,IFL = infrrrow liquid' bDR = Damagerating scale 0 - 10. "meanswith sameletter are not significantlydifferent at P < 0'05' Abundantchinch bugs resulted in severedamage^to sorghum^seedlings at Galva (Table 8). At Location l, sJrghumtreated with Gaucho@or Ponchot was significantly iess damagedby chinch bugs comparedto the nontreatedcheck, and Poncho* was = significantiymoie effectivethan Gaucho@ = 2, F 274.9,P: 0'0001).At Location2' ldf = = soighumtreated with Cruiser@or Gaucho@also was sigrificantly (!f 2' F-= 42.1' P plotsbut did not differ O.O6Of; less damaged by chinchbugs compared to the nontreated from each other. Yield was not out"inea from sorghum at either location at Galva becausethe growerdestroyed the plots. '{ TABLE 8. Chinch Bug control on sorghrm with seedtreahnents, Galva, KS, 2002. DamageRating ab Treatrnent Location I location 2 Gaucho480 DK-54 250gal1000 kg 3.5b 0.5b Cruiser5 FS DK-54 200 gal1000kg 0.5b Poncho DK.54 2000ppm 2.3c Untreated DK.54 9.3a 9.5a "DR = Damagerating scale 0 - 10. omeans with iame letter arenot significantly different at P < 0'05' 220 All 2001treatrnents significantlyreduced greenbug abundance on 2 June(df: 19, F=14.56,P=0.0001)and6June(df=19,F=8.35,P=0.0001).Significantdifferences in greenbugabundance also occurredamong the four sorghumhybrids tested (Table 9). In July, greenbugshad increased.toas many as 561.0per plant on the most susceptible nontreatedhybrid; insecticide-treatedplots had considerablyfewer greenbugs,often sigrificantly fewer so (df = 19, F = 3.16, P = 0.0004).Yield drita w=ie not obtained becauseofdamage by birds. TABLE 9. Greenbug Control on Sorghum with Treated Seed and Soil-Applied tnsecticiaes,Uays, Greenbugsper planton o Treatment Hybrid " lEtmg4_ Hybrid FormForm" Rate 2 Jun 6 Jun 24 Jgl Untreated NC+ 271 8.3c 8.3c 336.5abc Gaucho48O NC+271 ST l.l ml/kg 0.rd 0.0d 84.5cd Cruiser NC+ 271 ST 2009/100 kg 0.0d 0.0d 3r.5d Untreated 560y I l.7bc 13.9ab 561.0a Gaucho480 560y l.l mlirkg 0.3d 0.0d 19.5d Cruiser 560y ST 2009/100 kg 0.ld 0.0d 60.3d Furadan NC+ 271 4F 0.2mVm 0.0d 0.0d 97.8cd Temik NC+ ZZt 15c 0.9kglha 0.0d 0.0d l53.8bcd Untreated DK-36 23.0a 18,5a l03.3cd Gaucho480 DK-36 ST l.l ml/kg 0.0d 0.0d l37.8cd Poncho DK-36 ST 2Q00ppm l.0d 0.ld 459.8a Clothianidin600 DK-36 ST 2000ppm 0.0d 0.0d 83.0cd Clothianidin600 DK-36 ST 2500ppm 0.0d 0.0d 4t.7d Clothianidin600 DK-36 ST 0.13ml/kg 0.4d 0.0d l00.3cd Gaucho480 DK-36 ST 0.2 ml/lrg 0.7d 0.0d 409.5ab Temik DK.36 l5G 0.9kg/ha 0.0d 0.0d I I l.3cd Untreated P8500 t4.5b l0.5bc l31.7cd Gaucho480 P8500 ST 250!r00kg 0.0d 0.0d 18.8d Cruiser5 FS P8500 ST 200g/100 kg 0.2d 0.0d 20.3d Temik P8500 15G 0.9 0.ld 0.0d 10.0d t ST = seedtreatments, F = flowable, = granular. " C meanswith sameletter are not significantlydifferent at p < 0.05. significant greenbugcontrol was found for all 2002 treatmontson 6 June when plantswere in the four-leafstage (Table l0). Therewas no significantdifference among seedtreatments (Poncho@, Gaucho@, and cruiser$ and indrow liquid and granulai = = insecticides(df 19, F 2.64,P : 0.0024).com leaf aphidswere abundanton some plants on 17 July. While more plants in nontreatedthan hlated plots were infestedwith more than 200 aphidsper plant, no significantdifferences among treatrnents were found becauseof the greatamount of variationin the abundanceof coir leaf aphidsacross the plots. 221 TABLE 10. Greenbugand Corn Leaf Aphid Control with Seedand Soil Treatments, 2002. Treatment Hybrid Form Place" Rate GB/plant" Plants/plot > 200 cLA on 6.!qqe on 17July Untreated K S 560y 12.5b 3.5a Gaucho480 K S 560y ST l.l ml/kg 1.0cd 0.8a Poncho K S 560y :" ST 2000ppm a 0.8cd 0.3a Lttt6 K S 560y ST 2000ppm a 0.3d 0.0a Temik K S 560y 15G IFG 0.9 kg/ha 0.0d l.3a Counter K S 560y 15G IFC 0.9 kg/ha ).)co 0.3a Furadan K S 560y 4F IFL 0.2mVm 5.3cd 0.0a Untreated DK 54 13.8b 3.0a Gaucho480 DK 54 FS ST 250 galrcO} kg seed l.8cd l.3a Cruiser DK 54 5FS ST 200 gal1000kg seed l.5cd 0.5a A9765 DK 54 FS ST 50 gal1000kg seed L8cd 3.0a L9765 DK 54 FS ST 100gal1000 kg seed 2.Scd 0.5a Counter DK 54 20 CG IF 5.6gal100 m 3.5cd 2.8a Furadan DK 54 4F IFL 0.2ml/m 6.Ocd 4.0a Untreated NC+ 271 22.5a 4.5a Gaucho NC+ 271 4FL ST 1.1mVkg 0.0d l.3a Cruiser NC+ 271 5FS ST 200 gal1000kg seed 0.5cd 0.3a Temik NC+ 271 I5 GR IFG 0.9 kg/ha 0.3d 2.0a Counter NC+ 271 15G IFG 0.9 kg/ha 6.5c 2.3a Furadan NC+ 271 4F IFL O.2mVm 6.3cd 3.3a u ST = seedtreatrnents, IFG = infurrow granular,IFL: infunow liquid' b meanswith sameletter are not significantlydifferent at P < 0'05' DISCUSSION AII threeseed-treatment insecticides evaluated, Gaucho@ (imidacloprid) Cruiser@ (thiomethoxam)and Poncho@(clothianidin), controlled chinch bugs and greenbugs' were These three seed-treatrnentinsecticides were equally effective. Small differences of detectedwhen various rates of the insecticideswere used' Because of the convenience pests(chinch seedtreatments, these products will be importantwhere one or more of the bug or greenbug)are chronic problems. In the testsinvolving imidacloprid and thiomethoxamon hybridsat six locations yield betweentreated during a three-yearperioa [ZOOO-200i),significant differences in locationsin and n6ntreated,o.ghu- occuned at threeoifive locationsin 2000, one ofsix insecticidal 2001, and on. of six locations in 2002. Sorghumyield in responseto frequently treatmentdiffered most often at Hesston(two of the three years), a,location in one infestedwith chinch bugs. Damagingnumbers of chinch bugs were detected.there treatments' of the two yearswhen sorghumyiitOs OiffefeOas a result of insecticidal in which Theseresults are similar tJ tno." of a prwious study (Wilde et al. 1999) relatedto the significantincreases rn yield of insecticid-etreited sorghumwere usually insecticidal pr".r.n., of someinr.ri p.tt. When sorgbumyield diifered in responseto Cruiser@or treatment, no significant'difference in yield t"f*."n plots treated with 222 Gaucho@was found, except at one site in one year. There were significant effects on standestablishment at five of six locationsin 2000,four of six in 2001and five of six in 20Q2.Bttt,lack of moisture at severallocations in 2001 and 2002 may have affectedthe yieldsofthe sorghumhybrids. ACKNOWLEDGMENT This is contributionNo. 04-001-Jof the KansasAgricultural Experiment Station. LITERATURE CITED Archer,T.L. lgg4. Economicinjury levelsand chemical control of Russianwheat aphid, pp.97-102.1nF. Pearis,M, Kroening,and C. Simmons[eds.], Proc' Sixth Wheat Aphid Workshop,Fort Collins,CO' Harvey,T. L., andH. L. Hackerofi.1969. Recognition of a greenbugbiotlpe injuriousto sorghum.J. Econ.Entomol. 62:776-779. Harvey,T. L., K.D. Kofoid, T. J. Martin, and P. E. Sloderbeck.1991. A new greenbug virulentto E-biotlpe-resistantsorghum. Crop Sci. 3 I : 1689-l 69I ' Pike, K. S., G. L. Reed,G. T. Graf, andD. Allison. 1993.Compatibility of imidacloprid with fungicides as a seed-treatmentcontrol of Russianwheat aphid (Homoptera: Aphididae) and effect on germination growth, and yield of wheat and barley. J' Econ.Enotoml, 86: 586-593. SASkrstitute. 1988. Stat user's guide. 6.03 ed. SAS Institute,Cary, NC. Sloderbeck,P. E., M. D. Witt, and L. L. Buschmann.1996. Effects of imidaclopridseed treatnent on greenbug (Homoptera: Aphididae) infestations on two sorghum hybrids.Southwest, Entomol. 2l : l8l-1 87. Spike, B. P., R. J. Wright and S. D. Danielson.1991. Chinch bug outlook. NebFact NF9l-31.Univ. Nebraska Coop Ext. Dir. Tharp,D., S. L. Blodgett,and G. D. Johnson.2000. Efficacy of imidaclopridfor control of cerealleaf beetle(Coleoptera: Chrysomelidae) in barley.J. Econ.Entomol. 93: 38-42. Wilde, G. 1997. Etrect of imidacloprid seedtreatment and planting time applicationsof insecticideson chinch bug (Heteroptera:Lygaeidae) and resulting yields of sorghum.J. Agic. Entomol.14: 385-391. Wilde, G., K. Roozeboom,M. Claassen,P. Sloderbeck,M. Witt, K. Janssen,T. Hawey, K. Kofoid, L. Brooks, and R. Shufran. 1999. Does the systemicinsecticide imidacloprid(Gaucho) havea direct effect on yield of grain sorghum?J' Prod. Agnc.12:382-389. Wilde, G. E., R. J. Whitworth,M. Claassen,and R. A. Shufran.2001. Seed treatnent for controlof wheatinsects and its effecton yield. J. Agric. UrbanEntomol. 18: l-11. 223 vol.29 NO.3 SOUTHWESTERNENTOMOLOGIST SEPT.2OO4 REMOTE SENSINGTO DETECT PLANT STRESS,WTTITP-ARTICTJLA+ REFERENCETO STRESSCAUSED BY THE GREENBUG,:A REVIEW, ZhrmingYang, MaheshN. Rao, S. DeanKindlef , andNorman C. Elliott3 Centerfor the Applications of RemoteSensing, Department of Geography,Oklahoma StateUniversity, Stillwater, OK 74078 ABSTRACT Crop stresscaused by adversegrowing conditions,such as nutrient deficiency,pest infestation, disease,and drought, can be detectedusing techniquesof remote sensing. Symptomsof crop stresscan be detectedby variation in reflectancefrom plant surfacesof incident solar radiation in particular wavelength bands using radiometry. For example, someradiometers can record the quantity oflight reflectedin red and near infrared bands, which has proven useful for detectingplant stress. Vegetationindices calculatedfrom the quantity of reflected light in thesebands have been used to quantifr levels of stress. Plants subjectedto certain stresses,for example water stress,have higher leaftemperaturethan non-stressedplants. A change in the amount of thermal energy radiated by plants can sometimes be detected using infrared thermometers. In this review, applications of radiometryfor detectingplant stress,with particular emphasison detectingstress to cereals causedby the greenbug,Schizaphis graminum Rondani, are summarizedand approaches for future researchare ouflined. INTRODUCTION The Greenbug,Schizaphis graminum Rondani, is an important pest of wheat, sorghum,barley and other cereals. Localized or widespreadinfestations by the greenbug occur in almost every year in the Great Plains of the United Statesand causesignificant economic impact on cereal production, especially on wheat and sorghum @etersel al. 1997,Webster et al. 2000). Eachyear largequantities of insecticideare usedto control greenbuginfestations in winter wheat (Knutson and Ree 1998). To decreaseeconomic lossesand minimize adverseenvironmental impacts, timely and precisedetection of stress to cereal crops causedby greenbuginfestation is necessaryand important for farmers' Timely control dependson early detectionofgreenbug infestationsin fields, wtrich could be enhancedusing techniques of remotesensing. The purposeof this reviewis to examine applications of remote sensing,particularly spectroscopyand radiometry, for crop stress detection,with particular emphasison detectinggreenbug infestations in cereals. Basedon the review, researchneeds for remotesensing to detectgreenbug infestations are identified. IHomoptera:Aphididae 'Disclaimer: Mentionof tradenames or commercialproducts in this articleis solelyfor the purposeof providing specific information and doesnot imply recommendationor endorsementby the U.S. Departrnentof Agriculture. 'USDA-ARS PlantScience Research Laboratory, l30l N. WesternSt., Stillwater, OK 74075 227 PLANT STRESS Plant sftess is defined as a significant deviation from the optimal conditions for plant growth that could causeharmful effects when the limit of planis' ability to adjust is reached(Larcher 1995). Plant stress can affect almost part every of a plant,'stess, although typically one or a few plant structuresare influencedduring the early stages-of Lelaf responsesto different stresses.ue very important when considering remote sensing techniquesused to detectcrop stress. When the water contentof plant iells is lower than optimum and causesa degreeofmetabolic disturbance,a plant is said to be sufferingwater stress(Fitter and Hay 198 I ). Leaf curling, wilt, or reducedleaf areaexpansion are general symptomsof water stress(Alscher et al. 1990). The extentof impact of stresson plant leavesdepends on the occurrenceofwater stressrelative to the phenologicalstage olthe plant and severity of the water deficit. Nitrogen deficiency is a common nufient deficiency found in plants. When the requiredamount of nitrogen is not available,plants are said to be under nitrogen deficiency or nitrogen stress(Larcher 1995). plant Gaves generallytum yellow (overallchlorosis) under nitogen stress.In youngplants the whole plant turns yellow while in older plants the deficiency is more pronouncedin older leaves (Archer 1988). Insectinfestation refers to the presenceof large numbersof insectson plants and the injury to plants causedby theseinsects. Changein leaf color or leaf areaare coftrmon symptoms of insect infestation, and some changesinvolve cellular and tissue deteriorationleading to leafagingand death (Fogal et aL.1997). Greenbugsfeed in colonies, usually on the undersidesof leaves,where they suck plant sap and inject a toxic substanceinto the plant (Royer et al. 1998). Symptomsof injury to plants causedby greenbuginfestation initially appearas groupsof small, reddish, pinpoint spotson infestedleaves. Later, as feedingcontinues, leaves tum yellow and begin to die (Royeret al. 1998). Damageto the crop is relatedto numberof greenbugsand the length oftime they persist on the plants. It also dependson plant size, vigor and growth stage,and moistureconditions. Plantsinfested by greenbugsalso showphysiological and metabolic changes,such as a decreasein water potential and chlorophyll, and lower rates of COz assimilation(Cabrera et al. 1995). The injury causedby insects,such as the greenbug,can appeaxsuperficially like injury causedby other stresses,such as water or nitrogen stress,which can confound efforts to identifr greenbuginfestations using remote sensing(e.g., Michels et al. 1999). REMOTE SENSINGTO DETECTPLANT STRESS Image-basedremote sensing,such as by airborne and satellite systems,has been successfullyused in detectionof plant stress,for exampleinsect infestationon grapes (Hugh-Joneset al. 1992,Bell 1995,Royle and Lathrop 1994, Maas et al. 1999). However, technical limitations aflect its application for crop stress detection, with perhaps the greatestlimitation being instrumentdesign. Currently, satellite-basedsensors have coarse spatial resolution, which may not be adequateto detectspatial variation in plant stressat the scale of its occurrence within an agricultural crop. For aircraft-basedsensors, calibration and geometriccorrection are often difficult for large area coverage(Moran et al.1997). Ground-basedspectroscopy and radiometryhas beenwidely usedin crop stress detectionbut maynot be practicalfor implementationat a field scale(Hatfield 1990). Whether accomplishedby airborne or ground basedplatforms, all remote sensing systems for detecting plant stress rely on measuringdifferences in light reflectanceor emittancebetween stressed and healthy plants. Reflectanceoflight by leavesis governed by leaf surface properties, internal skucture, and the concentrationand dishibution of biochemical components, such as chlorophyll and other photosynthetically active molecules,and water. As a result, remote sensinganalysis of reflectancecan be used to 228 assessthe physiologicalstatus ofplants @enuelaset al. 1993, 1997;Penuelas and Inoue 1999). As with all physicalor biologicalobjects, plants reflect incident sunlight according to a characteristicpattem. Plantstypically absorb a majorityof incidentlight in the visible wavelenglhsfrom approximately450-680 nm for use in photosynthesis(Fig. lA), and stronglyreflect light in the near-infraredwavelengths from 750-1300nm (Fig.1B). A B-Carotene 0.01l0 38{} 450 495 570590 620 Wrvolength(mm) Rell*etrnce a NIR Refleqtance Brxrd \ ll I I {50 550650?3{1810 tss t0s0ll5$r!5{, l3gr l{5r}t5J,0l6t0 t?g) tt30 t95020502ts} Wnvebngth (mm) FIG. l. A) Absorption bandsfor incident solar radiation for various plant photo-pigments; B) Reflectanceof incident solar radiation by plants, showing the dominant near-infrared (NIR) reflectanceband. Adaptedfrom http://www.dasnr.okstate.edu/nitrogen_use/sensor_ based_improvement_olnue.htm. 229 Plants under stressexhibit a decreasein reflectancein the near infrared portion of the spectrum,and reducedabsorption of light in the photosyntheticallyactive portion of the spectrum,with a consequentshift in the so-calledred edge(Shibayama et al. 1993, Malthus and Madeira 1993, Carter 1993). Therefore,reflectance can be usedto derive indicatorsof crop conditionsand to assesslevels of stress(Fernandez et al. 1994). Plants subjectedto water stresshave higher leaf temperahre than normal plants. Other types of crop stressrelated to water uptake by plant roots or translocationof water to leavesfor evaporationalso result in higherleaf temperafures.As a result,a changein the amountof thermal energy radiated by plants under water stress can sometimesbe detectedusing infraredthermometers (Pinter 1979, Michels et al. 1999). Radiometryyields quantitativemeastrements of radiance,irradiance, reflectance or transmissionby objects. These instrumentsmeasuxe both inadiance and radianceof the plant,which canbe correlatedwith the biological,chemical, and physicalattributes of the plant. Radiometersare often hand-held,mounted on a tripod, ladder,scaffolding, or tall building, or in aircraft or satellites. It is sometimespossible to control the monitoring conditions using radiometry, and measurementscan be quantified and repeatedunder the same or similar conditions. Radiometry allows for precise analysis and interpretation because the crop can be sampled directly to measure the properties affecting leaf reflectanceor transmission(Goetz and Srivastava 1985). Remote sensingto detect nitrogen and water stresshas receivedconsiderable research attention. Multi-spectral radiometers and infrared thermometershave been successfullyused to detectand quantifr nitrogen deficiencyin agriculturalplants (Vouillot et al. 1998,Blackmer et al. 1994,Sembiring et al. 1999). Variouswavelengths of reflected light have beenused to detectnitrogen deficiency in crops. For example,red and near infrared wavelength bands were used for nitrogen stress detection in winter wheat (Vouillot et al. 1998,Sembiring et al. 1999),but a yellow bandcentered at 550 nm andan infrared band were used for corn (Blackmer et al. 1994). The Normalized Difference VegetationIndex (NDVI) is often used for plant stressdetection. NDVI is calculatedas follows: NDVI: (NIR - Red)/ (NIR + Red), where Red and NIR are reflectance in the red and near infrared wavelength bands, respectively(Lillisand and Kiefer 1987). Similar quantities,such as greenNDVI, where red light is replacedby a green wavelengthband have also been shown to have value as indicatorsofplant condition(Vouillot et al. 1998,Sembiring et al. 1999). Waterstress has been studied using spectroscopy and radiometry, often using hand- held multi-spectralradiometers (Ripple 1986,Mahey et al. l99l). Studieshave also been done to detect water stressin plants using hyper-spectralspectoscopy (Shibayamaet al. 1993;Fernandezetal.1994;Penuelasetal.1993,1997).Thewavelengthbandsusedto measurewater stressin wheat have varied among sfudies from different crops. It was found that red and middle infraredbands were sensitiveto water stressin snapbean,red and near infrared bandswere sensitiveto water stresson wheat, and a near infrared band centeredat 960 nm was identified that was sensitivefor rice (Ripple 1986, Mahey et al. 1991,Shibayama et al. 1993). Carter(1991) reported that reflectancein visible wavelengthbands from 535-640 nm and 685-700nm weremost sensitivefor detectingwater stress in plants,but Penuelas et al. (1993, 1997)found that changein reflectancein a nearinfrared band from 950-970 nm was the most effective for detectingwater shess.Variable results may be causedby differencesin the anatomicalstructure and water content of leaves from different plant species,which may alter reflectancepatterns. Indices involving multiple wavelengthbands have been used for detectingwater stress. Among them NDVI has beenwidely used(Shibayama et al. 1993,Mahey et al. 230 1991,Fernandez et al. 1994),although the specificwavelengths used to calculateNDVI differed among studies. In addition, a water index was developedand usedto detectcrop water stress(Penuelas et al. 1993,Penuelas and Filella 1997,Riedell and Blackmer 1999). The water index is calculatedas, waterindex : R970/ R900, where R970 and R900 are reflectancein bandscentered at 970 nm and 900 nm. Foliage temp€rafirxecan be incorporatedinto indices, such as the crop water sfressindex (CWS| (Hatfield 1990). Detection methodsare basedon comparingfoliage and air temperature andCWSI is calculatedas, cwsl: [(Tr- TJ - Gf - Ta)]4(T1-T3)u - (T6- T)1, where T1 and T" reprcsentfoliage and air temperature, (Tr - Ta)l representsthe lower baselineand (Tr - T")orepresents the upperbaseline (Idso et al. I 98I ). Infrared thermometersare frequently usedto monitor foliage temperatureto detect water stess (Stark and Wright l9&5,Yazat et al. 1999,Carcova et al. 1998,Alves and Pereira 2000). Comparedto water stressdetection by measuringleaf reflectanceusing multi-spectral radiometers, measurements of leaf temperatures using infrared thermometers,and indicessuch as the CWSI havebeen widely used. This maybe because of the relatively low cost of measurementequipment and the availability of standardized techniques. REMOTE SENSINGTO DETECT GREENBUGINFESTATIONS Deol et al. (1997)and Ma et al. (1998)demonstrated in sorghumthat injury caused by greenbugscan be detectedindirectly by measuringthe chlorophyll content of leaves infestedby greenbugs.In a laboratorystudy, Riedell and Blackmer (1999) used a portable ASD spectroradiometerto determinethe wavelengthsof light reflectedfrom wheat leaves that were most sensitiveto greenbuginfestation. Reflectancefrom detachedwheat leaves infested with greenbugs wns mensured for wavelengths from 350-1075 nm at approximately about 1.4 nm intervals. Normalized total pigment to chlorophyll index (NPCD and the water bandindex were computedfor analysis. NPCI is calculatedas, NPCI =(R680- R430yR680+ R430, where R680 and R430 are reflectancein bands centeredat 680 nm and 430 nm. and the water bandindex is, water bandindex = R950/R900, where R950 and R900 are reflectancein bandscentered at 950 nm and 900 nm (Penuelas et al.1993,1997). Total leafchlorophyllconcentration and chlorophylla./b ratios ofleaves fed on by greenbugswere significantly lower than those of healthy leaves. Reflectancein the 625- 635 nm and 680-696 nm ranges and the normalized total pigment to chlorophyll index were sigrrificantlycorrelated with total chlorophyll concentrationsin infestedleaves, which was reducedby greenbugfeeding. Michels et al. (1999) used infrared thermometersto monitor the temperature difference of wheat plants grown in flats in a greenhouseunder four regimes:normally- watered and not infested, normally-wateredand infested, water-stressedbut not infested, and water-stressedand infested. The experimentdemonstrated a significant temp€rature difference between non-infested and greenbug infested wheat, and the difference in temperatureincreased with the abundanceof greenbugs. However, no difference in temperahre between normally-wateredbut greenbug infested plants and non-infested, water-stressedplants was detected,indicating that infrared thermometermeasurements were not reliable for diflerentiating betweengreenbug intbstation and water shess(Michels et al. 1999). The studiesmentioned above regarding greenbug injurywere all conductedin the laboratory or greenhouse. Although additional research is needed before firm 231 conclusionscan be drawn,it appearsthat leafreflectanceis a betterindicator ofgreenbug stressthan leaf temperature. Greenbug outbreaksoften appear at times when the wheat crop is under water stess (Ortrnan and Painter 1960; Michels and Undersander1986, Michels and Behle 1998). Michels and Undersander(1986) observedthat the number and distribution of greenbugson sorghum plants was strongly affected by water stress. Thus plants uncler water stresswere more sensitiveto greenbuginfestation than non-waterstressed plants, It was found that the combination of water stressand greenbugstress did not causemore decreasesin factors including C assimilation rate and total leaf chlorophyll content on resistantplants over those causedby water stressalone (Ryan et a1.1987). In addition, Cabreaet al. (1995) reportedthat greenbuginfestation and drought-inducedwater shesson barley produced similar decreasesin relative water potential and content, and leaf chlorophyll content. Thus, it has been diflicult to differentiatewater stressfrom greenbug infestation. Consideringtheir frequent co-occurrencein the field, it may be diffrcult to differentiate these two types of stressesunder the field conditions. However, this assumptionhas yet to be tested. Different stressesmay often co-occurin cerealsgrown under field conditions. If it is desired to find wavelength bands diagnostic of greenbuginduced stress, it will be necessaryto differentiate water stress and other conmon stressesto cereals from that causedby greenbuginfestation. Severalvegetation indices, such as NDVI, havebeen used for crop stressdetection, which could be investigatedfor their potential to differentiate various factors that causestress. For example,the YellownessIndex mezuuresthe extent of chlorosisof leavesin stressedplants (Adams et al. I 999),and is calculatedas, yellowness: R(f.r) - 2R(f"0)+ R(1.+r)l/ AI2, whereR(X.e) is the reflectanceat the centralwaveband, R(},-l) and R(l*r) are the lower and higher wavebands,Al" is the spectraldistance between wavelengths (Af : i.0 - l'-r : Lr - la). Yellowness is not affected by leaf structureor water content and thus may be useful for differentiating greenbuginfestation from other cornmon stesses. Investigationsto examinethe value of various vegetationindices for detectinggreenbug infestation could be enlightening. The use of hyper-spectralremote sensing data may be of value for this purpose. The size of the minimum measurementunit, usually called the grain size, typically controls what can be observed within a particular spatial area using a particular measurementprotocol. The processescreating spatial pattern at a particular spatial scale usually depend on the scale of observation. tn particular, the size of the minimum measurementunit will place constraints on what can be observed and what appears importantin measuringa natural phenomenon(Wiens 1989). Pixel size may vary for particular multi-spectralor hyper-spechalremote sensing systems. With respectto the use of remote sensingto detect crops stress,it seemsreasonable to expect that reflectance pattems will vary with the gtain size (pixel size) of the measuringdevise. In wheat for example, Riedell and Blackmer (1999) identified wavelengths sensitive to greenbug infestation in wheat using measurementmade at the scale of individual leaves. Measurementsmade in the freld from airbome or ground basedplatforms would measure reflectancefrom the plant canopy. Such measurementswould involve reflectancefrom injured wheat leavesaccording to the spectralpattern identified by Riedell and Blackmer (1999), but would also involve reflectancefrom soil, from plant structuresother than leaves,from healthyleaves, and leavessubjected to otherstresses. Also, the angleofthe reflective surface plant structures relative to the direction of incoming solar radiation would affect the amount of light reflected to the imaging system. To date, it has not proven possible to identiff unique spectral characteristicsof greenbugiqiury to wheat plants that differentiate it from other factors causing stress(Michels et al. 1999; Yang, 232 unpublisheddata). Whetherthe spectralsignature of leavesinjwed by greenbugscould be diflerentiated from amongthe many other signalswill require detailedfield investigations using multi-sprectraland perhapshyper-spectral imaging systems. An alternative to developing a "spectral signature" for detecting greenbug "spatial infestationsis to developa signature". Greenbugpopulations in wheatfields grow in a characteristic way. Greenbugsinitially establish at locations in a field due to colonization by aerially dispersingindividuals, and increasein density in theseinitial sites to form local patchesof high greenbugdensity that gradually increasein size. As the population grows, the local patchesbegin to coalesceto form a more complex network of patches@obert 1987). This pattem of colonization, population growth, and spreadmay result in characteristic spatiat disributions of greenbugsin wheat fields @lliott and Kieckhefer 1987, Elliott et al. 1994), which may be diagnostic. Migrant greenbugs traveling long distancescan colonize a wheat field, or greenbugscan fly from nearbyfields to colonizea field (Medler and smith 1960,Loxdale et al. 1993),and the dishibutionof colonizers:tcross a field might differ for thesetwo scenarios.Even thoughthe distribution of colonizersin the field migbt differ, the growth of local patchesestablished by colonizing greenbugsover time would still occur, and may be identifiable in remotely sensedmulti- spectralimagery. 0.90 o 0.89 3' Y=0.88- 0.0024x 3 r'o oa' a f = 0.36 {-r! a 0.87 $io a o E ata .O a z o oa o o No. Greenbugs/ Tiller FlG.2. NDVI versusthe density of greenbugsin l-m2 plots in a wheatfield near Perkins, oklahoma. Datawere acquiredon November6,2001using an ssr cRISo muhi-specnal imaging system. The existenceof diagnosticspatial patterns of plant stresscaused by greenbugshas not beeninvestigated. However, field testshave demonstratedthat NDVI calculatedfrom red and near infrared bandstypical of thosefound on earthobservation satellites and other airbome remote sensingplatforms can be used to detect greenbuginfested wheat in the field (Fig. 2) (NCE, MN& SDK, G. J. Michels,K. L. Giles,and D. A. Waits,unpublished data). 233 CONCLUSIONS Progresshas been made at detecting greenbuginfestation in crops using remote sensing of reflected and emitted electromagneticradiation. However, researchon the subject is still incomplete. Since water stressand other plant stressescan co-occur with greenbuginfestations, research should focus on methodsto differentiate vmious stresses from greenbug-inducedstress under field conditions. Two possibilities for achievingthe goal of using remote sensing for monitoring greenbug infestations in IPM programs include the use of multi-spectral or hyper-spectralimaging systemsto identif, sensitive wavelengthsthat distinguish stresscaused by greenbugsfrom other types of stress,and using such systemsto detect spatial pattems of plant stress, which are diagnostic of greenbuginfestation. In either case,follow-up samplingwithin the field would be required to determineif the greenbuginfestation exceededthe economic threshold for insecticide treatnent. ACKNOWLEDGMENT We thank Tim Johnsonfor creatingthe figures displayedin the paper,and David Waits, Frank Schiebe,and Monte Stewartof SST DevelopmentGroup, Inc. for providing accessto and assistancewith the useof the SST CRIS imaging system. LITERATURECITED Adams,M. L., W. D. Philpot,and W. A. Norvell. 1999.Yellowness index: an application of spectralsecond derivatives to estimatechlorosis of leavesin stressedvegetation. Int.J. 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Econ.Entomol. 82:439-444. Michels, G.J., Jr., G. Piccinni, C. M. Rush, and D. A. Fritts. 1999. Using infrared transducersto sense greenbug (Homoptera: Aphididae) infestations in winter wheat.Southwest. Entomol. 24: 269-279. 235 Moran,M. s., Y. Inoue,and E. M. Bames. 1997. opportunitiesand limitations for image- basedremote sensing in precisioncrop management.Remote sensing Environ. 6l: 319-346. ortnan, E. E., and R. H. Painter. 1960. euantitative measurementsof damageby greenbug, Toxoptera Graminum, to four wheat varieties. J. Econ. Entomol. 53: 798-802. Penuelas,J., I. Filella, c. Biel, L. serrano,and R. save. 1993. Thereflectance at the 950- 970 nm regionas an indicatorofplant waterstatus. Int. J. RemoteSens. l4:lggz- 1905. Penuelas,J., J. Pinol, and I. Filella. 1997. Estimationof plant waterconceirtration by the reflectanceWater Index WI (R900/R970).Int. J. RemoteSensing. 18:2869-2875. Penuelas,J. and Y. Inoue. 1999. Reflectanceindices indicative ofchanges in waterand pigmentcontents ofpeanut and wheat leaves. Photosynthetica. 36: 355-360. Peters,D. C., F. Ullah, M. A. Karner,W. B. Massey,P. G. Mulder, andV. H Beregovoy. 1997. Greenbug(Homoptera: Aphididae) biotype surveysin Oklahoma,l99l- 1996.J. KansasEntomol. Soc. 70: 120-128. Pinter, P. J. 1979. Remote detection of biological stressesin plants with infrared thermometry.Science. 205 : 585-587. Riedell, W. E. and T. M. Blackmer. 1999. kaf reflectancespectra of cereal aphid- damagedwheat.Crop Science 39: 1835-1840. Ripple, W. 1986. Spectralreflectance relationships to leaf water stress.Photogr. Eng. RemoteSensing 52: 1669-1675. Robert, Y. 1987. Dispersionand migration. pp.299-314. In A. K. Minks and P. Harewijn (eds.),Aphids: Their Biolog, Natural Enemies,and Control. Yolume 2A. Elsevier SciencePublishing Company, New York. Royer,T. A., Giles, K. L. and Elliott, N.C. Small grain aphidsin Oklahoma OSU Ext. FactsF-7183. 1998. Royle, D., and R. Lathrop. 1994. Using Landsat Thematic Mapper data and change detectiontechniques to monitor hemlock forest health.Forest Science 43 : 327-335. Ryan, J. D., R. C. Johnson, R. D. Eikenbary, and K. W. Dorschner. 1987. Drought/greenbug interactions: photosynthesis of greenbug resistant and susceptiblewheat. Crop Science27:283-288. Sembiring,H., W. R. Raun,G. V. Johnson,M. L. Stone,J. B. Solie, and S. B. Phillips. 1999. Detection ofnitrogen and phosphorusnutrient statusin winter wheat using spectralradiance. J. PlantNutrition2l: 1207-1233. Shibayam4M., W. Takahashi,S. Morinaga,and T. Akiyama. 1993. Canopywater deficit detection in paddy rice using a high resolution field spectroradiometer.Remote SensingEnviron. 45: 117-126. Stark,J. C. and J. L. Wright. 1985. Relationshipbetween foliage temperature and water stressin potatoes.American Potato Journal 62:57-69. Vouillot, M. O., P. Huet, and P. Boissard. 1998. Early detectionof N deficiencyin a wheatcrop usingphysiological and radiometric methods. Agronomie l8: 117-130. Webster,J. A., R. Treat, L. Morgan, and N. Elliott. 2000. Economic impact of Russian wheat aphid and green bug in the westem United States 1993-94,1994-95,and 1997-98.USDA, Agricultural ResearchService Report, PSWCRL Rep. 00-001, Plant Scienceand Water ConservationResearch Laboratory, Stillwater, OK. Yaza4 4., T. A. Howell, D. A. Dusek,and K. S. Copeland. 1999. Evaluationof crop waterstress index for LEPA inigatedcom. LrigationScience l8: l7l-180. Wiens,J. A. 1989. Spatialscaling in ecolory.Functional Ecology 3:385-397. 236 I vol".29NO. 3 SOUTHWESTERNENTOMOLOGIST SEPT.2OO4 INSTRUCTIONS FOR PREPARATION OF MANUSCRIPTS Manuscripts that report results of routine laboratory or field experiments for which the primary purpose is gatheringbaseline data or reporting the results of a continuousscreening progran such as preliminary pesticidescreening hials, specieslists with no supportingbiological data, or preliminary host plant resistancet€sts are not acceptable. However, repirts of Jxperinients with insecticides, acaricides, and microbials are acceptable for the journal if they are comprehensiveand are related to economics,resistance, toxicology, or other broad subject areas. Bibliographies will not be published in ^9oalrwesternEntomologist. Scientific Notes can pertain to observationson new hosts, geographicrecords, and new techniques. Manuscript preparation and review are the sameas for regular articles. 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Si los nombresde insectosson mencionadosen el titulo (comritro scientifico),se debenin dar los nombres de orden y la familia en nota de pie de p6gina. Los nombrescientlficos se deber6n escribir con letrasit6licas en todo el manuscrito. 17. Todas las abreviaturas,etc., deber6najustarse al AIBS Style Manual for Biological Journals. 241 vol.29 NO.4 SOUTIIWESTERNENTOMOLOGIST DEC.2004 SEASONAL ABUNDANCE OF RICE ROOT APHID' IN WHEAT AND ITS EFFECTON FORAGE A}ID GRAIN YIELDS DeanKindler, Louis HeslEl, Norm Elliott, Tom Roy#, and Kris Giles3 USDA-ARS, Plant Scienceand Water ConservationResearch Laboratory l30l N. Westem St.,Stillwater, Oklahoma 74075. ABSTRACT Aphid seasonalabundance in wheat, Triticum aestivumL., was studied over a two- year period in cenfral Oklahomawith emphasison the field abundanceof the rice root aphid, RhopalosiphunruJiabdominalrs (Sasaki). The rice leafaphid,R. ruJiabdominalis;com leaf aphid,Rhopalosiphum maidis (Fitch); oat bird-cherry aphid,Rhopalosiphum padi (L.); and greenbug,Schizaphis graminum (Rondani), colonized winter wheatin Oklahomaduring the autumnsof200l and 2001. During eachofthe two years,rice root aphidsinfested winter wheatsoon after emergenceand continuedto increasein numberon the fall seededcrop until mid-December,at which time populationspeaked and then began to decline, so that aphidswere difficult to find by earlyJanuary. Rice root aphidpopulations of 3.6 aphidsper tiller at the end of a 60-day infestation period significantly reduced the forage yieldof wheat,which can result in a significant economicimpact since approximately67n/o of the 6- 7 million acresof winter wheat grown each year in Oklahoma is grazedby cattle. Grain yield wasnot significantlyreduced by a 60-dayrice root aphidinfestation. INTRODUCTION - fhere.are 27 speciesof aphidsthat utilize cultivated cerealsas warm-s€asonor year- around hostsin the United States@ikeet al. 1990). Someare widely distributedspecies that a^repr,esent annually and rqiure.cereal crops during the growing ,r*on; others are only sporadic Plsts or are only occasionallyfound or cer""t .tops. Some ofihese speciesari cryptic and their presenceon cerealcrops is overlookedexcept when severeoutbreaks occur. For the most part there is very little pubiishedinformation about aphidsin this latter group. potentially - _ _A important species of which little is icnown is Rhopalosiphum ruJiabdominalis (Sasaki),the rice root aphid. This aphid specieshas beenobserved feeding extorsively on cultivatedcereals (Robinson and Hsu-1963, Royer 1999,crr"pin et al. 2001) and may cause economic although losseshave apparentlynluo 6* quantified (chapin {am1qe, et al. 20Ql; s. Halbert, Florida Department or'egricutture, personal communication), The rice root aphid has a world-wide distribution @ia*man aod E^top 1984)' h North America, the rice root aphid has beenreported on various hostsin 26 states 'Homoptera:Aphididae 'usDA-ARs, Norttrem Grain InsectsResearch Laboratory,2923MedaryAve., Brookings, SouthDakota 57006. 'Dep.- Of Entomology& plant patholory, OSU, Stillwater, OK74O7L. 245 and Canada(Kieckhefer and Gustin 1967,Jedlinski l98l). Primaryhosts of the rice root aphid are Prunus species @oncaster 1956), whereas secondary (altemate) hosts are numerousspecies of Graminae,Clperaceae, and dioots, especiallypotato and tomato @atch 1938,Blackrnan and Eastop1984, Tsai and Liu 1998). The similar appearanceofthe bird cherry-oat qhtd, Rhopalosiphum padi L, and the rice root aphid often lead to misidentification, Damage to ceredls often attributed to the bird cherry-oat aphid may sometimesbe causedby the rice root aphid. A key distinguishing feature is that the bird cherry-oataphid has six antennalsegments whereas the rice root aphid only has five antennal segments@ike et al. 1990). Other non-diagrrosticcharacteristics can also be usedto separate the species, including the presence of dense setae on the rice root aphid, and slight differencesin coloration. The rice root aphid may be a particularly importantpest of cerealsin the GreatPlains (S. Halbert, Florida Departmentof Agriculture, personalcommunication). It may transmit barleyyellow dwarfdisease(Jedlinski 1981, Paliwal 1980,Gray et al. 1998)in additionto causing direct feeding damage. This aphid also nay occur on forage grasses,causing economic damageto them. We recently completed a one-yearpreliminary study on the seasonalabundance of the rice root aphid on fall plantedwinter wheat in central Oklahoma (SDK unpublisheddata). The rice root aphid was the most abundantaphid speciescollected dwing Septemberthrough October, with populations gradually decreasingto low levels in January. Also, one of us (LHS) had been askedto examinewinter wheat fields in central South Dakota tlat were heavily infestedwith cerealaphids in the fall of 1997and 1999,and a predominantaphid in these fields was the rice root aphid (unpublisheddata). The early infestation of wheat by the rice root aphid suggeststhat it has the potential to reduceforage production of wheat, which is widely grazedby cattle most years in Oklahoma and other statesin the GreatPlains. The discoveryof rice root aphidsassociated with cultivated wheat in the GreatPlains in recentyears and resultsof our preliminary study indicatethat fiuther researchis warranted to determinethe importanceof the rice root aphid as an economicpest of cultivated cereals. We conductedtwo studies to obtain insight into the economic importanceof the rice root aphid in Oklahoma. The first was a two-year field study to determine the seasonal abundancepattems of the rice root aphid and other common cereal aphids in production winter wheat fields in central Oklahoma. The secondwas a laboratorystudy to determinethe effectsofrice root aphidinfestation on the forageand grain yields ofwinter wheat. MATERIALS AND METHODS To determinethe abundanceof aphidsin winter wheat in Oklatrom4 we sampled10 randomly selectedwinter wheat fields in Garfield County, Oklahoma,during the 2001-2002 growing season. Samplingconsisted of visiting eachfield biweekly beginningsoon after the emergenceof the fall planted wheat and continued into May by which time the wheat had senesced. Samples from fields were taken using a spadewith a six-inch wide blade by placing the blade next to wheat plants within a row of wheat and digging below the plants to remove the plants, their roots, and associatedsoil within the width of the blade. A single sample was taken fiom each field on a particular sampling occasion. Each sample was placed carefully into a zip lock plastic bag, kept cool, and returnedto the laboratory. During 2002-2003,a single field in PayneCounty, Oklahoma,was sampledbiweekly by taking l0 sampleswith the spadefrom randomly selectedlocations in the field' At the laboratory,each sample was placedinto a Berlesefunnel to extractthe aphids from the soil and plant sarnple. All aphids were identified to speciesand counted. In addition, the numberof plants and the numberof tillers in eachsample was determined. 246 Rice root aphidsused in this study were obtainedby collecting numerousindividuals from a winter wheat field near Brookings, South Dakota, in autumn 1999. We established non-viruliferous laboratory colonies by placing field-collected adult rice root aphids on sachetsof Parafilm@ (American National Can Co., Greenwich, Connecticut) membranes holding 20% (wt.) sucrosesolution and transferring their neonateoffspring to noninfested plants (Kieckhefer and Gellner 1992). Voucher specimensof the adult aphidsare deposited at the K. C. Emerson Entomology Museum, Oklahoma State University, Stillwater, Oklahoma. The aphids were cultured on wheat plants growing in l5.2cm diameter pots covered with cylindrical cellulose nifrate cages using methods describedby Starks and Burton (1977) wittr the following changes. First, the soil surface in the culture pot was coveredwith cedarchips. This apparentlyhelped to createa microenvhonmentat the baseof the plants, which was conduciveto aphid establishment,reproduction, and survival. Second, the aphidswere transfered to new cultureswhen alate forms beganto collect on the tops of the cages approximately 3 weeks after infestation. The transfer was accomplishedby exchanging the cages from an infested pot with that from a non-infestedpot. Several transfers could be made each day using this approach, until the original culture was exhausted. TAM-ll0 wheat seedwas vemalized in the refrigerator for 6 weeks at 385. The seed was planted in 48, 6-inch pots with one seedper pot. The pots were placed in two Puffer Hubbard plant gowth chambers(Kendro Manufacturing,Ashville, NC) at l8"c day and I loC night. Half (24 pots) were infestedwith rice root aphid and the other half (24 pots) were maintainedfree of aphids. The photoperiodwas maintainedat l0hr day and l4hr night. T\e 24 treatnent plants were infestedu/ith 25 rice root aphid per plant at 27 days,3Odays, and 34 days after planting to insure adequateinfestations on treatmentplants. Infestation was accomplishedby placrng cut plant material on the plants from rice root aphid culture pots. Plant growth chamberconditions were changedat 55 days after planting to promote plant tillering. The chamberterrperature was changedto lgoc day and l2'c night. The chamberphotoperiod was changedto I lhr day and l3hr night. At 63 days after planting, 12 infested pots and 12 non-infestedpots were removed and aphid numbersand forage yield componentswere determined. Specifically,the number of rice root aphids,height, weight (fresh and dry), and the numberof iillers were determined for eachplant. The roots were cleanedand dried before weighing._The remaining infested and non-infestedpots were treatedwith one-eighthtsp. of Marathon@lo/o granular insecticide (olympic Horticulh"ualProducts, co., Mainland, pA) per pot. The plants were then moved to a Rheem model 511-38 Environmental chamber (Rheem Manufacturing,Los Angeles, CA) maintainedat the temperatureand photoperioddescribed above. The ih""- chamber had a tall enoughinner chamberspace to allow plants to grow to maturity. Onceplants had grain 1{ure$, yield componentswere measuredfdr eachplant. Specifically, the numberof tillers, height,number offertile heads,number ofihfertile heads,number oiseeds per head, and seedweight were measuredfor eachplant. . The componentsof yield describedabove were statistically analyzedby analysisof variance using PROC GLM (SAS Institute 1990). Means comparisonswere accomplished using the Ryan-Einot-Gabriel-welschmultiple range test option in pRoc GLM. A single factor completely randomizedexperimental desigrrwas used with two levels of infestation with rice root aphids, un-infestedand infested,as the independentvariable, and a particular yield componentas the dependentvariable. RESULTS Rice root aphid, bird cheny oat aphid, corn leaf aphid, Rhopalosiphummaidis (Fitch), and greebros, Schizaphisgraminum (Ro4dani), werelQund infesting wheat during 247 fall in Garfield country, oK, wherefields were first sampledon g November2001 (Table l), lhe rice root aphid was the predominantaphid speciesmaking up 74.4%of the total aphidpopulation when the fields weresampled on 15 November20b1.- The rice root aphid continued to be part of the aphid fauna into 2002 where, on l l February, it represented 23.1%oof the total aphid fauna in the wheat fields. After 25 February 200i, the ptpulation of rice root aphidswas not detectedin samplesuntil 23 Lp/rl2002,wirenthey reappeared in small nurnbelsindicating that theyprobably overwintered in the fields. The iice ioot aphid, alongwith the bird cherryoat aphid,greenbug, and corn leafaphid werethe most abundant aplridspecies infesting the fields duringthe fall monthsof 2002. TABLE l. Compositeof Aphid SpeciesAbundance Infesting Winter Wheat During the 2001-2002Growing Season, Garfield, Co., OK. # Fields Total # Total # % oftotal aphids Total Aphids Date Sampled Plants tillers RRAffi tltS/200r 6 37.4 t7.3 30.2 15.1 t79 tr/ls/2001 6 74.4 0.4 24.6 0.6 rt/23/200t 9 338 22.4 ll.l 52.3 l4.l 566 t2/tll200t 9 8l 660 16.1 14.7 87.2 38.7 1004 r2/3r/200r 9 62 509 3.4 l.l 30.3 65.2 818 ur4t2002 9 63 474 5.8 0.0 47.7 46.5 155 t/2812002 9 74 677 t8.2 0.0 24.2 57.6 99 2nt/2002 9 104 647 23.t 0.0 23.t 53.9 l3 2/2s/2002 9 56 594 0.6 t.2 23.9 74.2 163 3nl/2002 9 50 560 0.0 0.0 41.9 58.1 43 3/26/2002 9 57 507 0.0 0.0 50.0 50.0 6 4/1012002 9 3l 361 0.0 0.0 39.7 60.2 88 4/23t2002 9 31 361 4.4 0.0 43.5 52.2 23 sl7/2002 9 l9 222 100.0 0.0 0.0 0.0 2 'Rice Root Aphid oComLraf Aphid 'Bird Cherry Oat Aphid oGreenbug Aphid speciesabundance and calculatedaphid daysfor the aphidsinfesting wheat in the fall of2002 in PayneCountn OII are presentedin Table 2. The rice root aphid infested the fall planted wheat soon after emergenceand continued to infest the wheat until 11 December 2002. The number of aphids and aphid days on each sampling date were generally greater for the rice root aphid than for the com leaf aphid, bird cherry-oataphid, and greenbug. Total aphid days for the rice root ryhrd Q0,226.5) were about 10 times greaterthan for the corn leaf aphid(1,159.5) and greenbug (1,011.0). The field was sampled for aphid abundanceon 30 October 2002 after 3.5 inches of rain had fallen on 27 October 2002. b spite of the rainfall, the rice root aphid still persistedin the field. Samplingwas 248 discontinued after 14 December 2002 becauseof the introduction of a large number of stockercattle to pastureon the wheat. TABLE 2. composite of Aphid SpeciesAbundance and calculated Aphid Days for the Aphid SpeciesInfesting Wheat in the Fall of 2002. PavneCo.. OK. Total No. orugqh,Aphid Spgcies# RRA per Aphid Days for EachSpecies" Date RRA" CLAb BCOA" GBd Tiller RRA CLA BCOA GB October4 160 0 0 0 2.76 October2l 93 0 ll 0 0.53 2024 0 88 0 October30 1068 50 8 38 2.94 5224.5 225 85.s t?l November2O 82 18 83 17 0.22 t2075 714 955.5 577.s Decemberll 4 3 3l g 0,01 903 220.5 rr97 262.s ToralAphids 1407 7r r33 63 -- 202265 rr5g.5 2326 l0lr oCorn Leaf Aphid Oat Aphid "Greenbug.Fird-Cherry 'Apryq days were calculated_by taking the difference between each sampling multiplying ' date and it by the meantotal aphidsoithe trro samplingdates . The comparisonof the forageyield compon€ntsof the wheatplants infestedand non- r-r9e^roor.anhidsarefreiented io iaure3. pranrrreighi w;noi innuencea nce33t-,"d.*Proot aphid infestation: uy H:1""u9t,dry root weight, dry prant dsht ;d th" totarnumber of tillers per plant were sigrificantly reducedwhei wheat plants were infestedwith meanof.50'3_aphids a final per prant or 2.56 a^phidsper tiller.d ;0 dq ili;*"". weigh!, dry plurt Dry root weight, and nwnber of titt"tr'ru"r" reducedby 52.9%, respectively. 42.3yoand 29.6%o, 3. JIBL! . _YTq Comparisonof ForageYield Components plants Infestedand Uninfested of TAM ll0 Wheat with theRice Ro-ot ft StandardEnor)". ForageYield Component %Differencefrom Uninfested Infested Contol No. aphids 0.0+ 0.00a 50.3+ 9.7Ib No. aphidsper tiller 0.0+ 0.00a 3.6+0.67b Plant height (cm) 62.6+ 1.36a 62.2+l.0ta -0.64 Dryrootwt. (g) 1.7* 0.10a 0.8+ O.OSU -52.94 Dryplahtwt. (g) 5.2+0.25a 3.0+ 0.13b 42.31 No. tillers 19.6* 1.06a 13.8+ 0.,14b -29.59 "Means within a colum" the Ryan-Einot-Gabriel-Welschmultiple ran;*;:- 249 The mean comparisonof grain yield componentsof wheat plants infested and non- infested with the rice root aphid are presentedin Table 4. The mean number of tillers and number of fertile headswere significantly reduced,while the number of infertile tillers was significantly increasedby the ilce root aphid. Alttrough mean plant height, the number of infertile heads,the numberof seeds,and seedweight were reducedwhen plantswere infested 'the with rice root aphids, means for each of these yield componentsw€re not sigrificantly different from the control. TABLE 4. Mean Grain Yield Componentsof TAM I l0 Wheat Plants Infested and Non- mfesteawittr lUce noot Grain Yield Grain Yield Component Component % Differe,ncefrom Contol Uninfested Infested Aphids/ tiller 0.00a 2.09b Plant height (cm) 73.9+0.64a 72.4+l.l6a -2.03 No. tillers 28.0+ 1.38a 24.1*0.78b -L3.93 No. fertile heads 16.5+ 1.29a ll.4 + Q.92b -30.91 -18.52 No. infertile heads 8.1+ 0.78a 6.6+ 1'0la No. infertiletillers 3.4*0.57b 6.1* 1.09a 44.26 -8.18 No. Seeds 345.8+ 18.62a 317.5+29.47a -12'35 Seedweight (g) 17.0+ 0.95a 14.9+ l '56a the Ryan-Einot-Gabriel-Welschmultiple rangetest' DISCUSSION pest The rice root aphid along with the bird cherry oat aphid and greenb}g are major speciesfound in wheai in Oklahoma. The rice root aphid was found infesting wheatin most areasof the state in the fall. The rice root aphid infested seedlingsin early fall soon after wheat emergenceand were presentuntil earlyNovemberwhen populationsbegin to decline' By mid Janiary populationswere low. A small numberof rice aphidsstill infestedwheat in laie April inoicatlng fte aphid is capableof overwinteringin Oklahoml (]aul9 l). The rice root apda impactedthe forage yield of wheat. An infestation of rice root aphids feeding for 60 days with a final population of 3.6 aphids/tiller reducedthe forage yield of a whiat ptantby lz.t% (Table 3). Field populationsreached nearly 3.0 rice root aphicts/ tiller by late Novemberduring our study (Table 2). The impact of suchpopulations on fo."g" yieldtould be substantialsiice about67%o of 7.|million acresof wheatis annually grazed-by'cattlein oklahoma (True et al. 2001). Grain yield componerntswere reduced,but iot sifficantly so, for infested wheat plants comparedto the cofiols in our laboratory .t ray ito" we simulatedthe approximatl length,cllle feedingperiod observedin the field. to lossiUty, the decline of the aphid feeding on wleat by early Janugrl allowed the wheat ...ou"iio* the feedingdamage before it was harvestedsometime in June. 250 The rice root aphid has only been recogrized the last few years infesting wheat in economic numbers (Royer 1999). In an earlier study, aphids were intensively collected throughout Oklahoma from 1958to 1964(Van Cleave 1970). Over 1,500individual samples were taken from all 77 countiesin the state. A total of40 generaand 68 speciesofaphids were recorded in the study, but only two samples,Stephens County, 16 March, 1960,and OkfuskeeCounty, October 19,1960,contained the rice root aphid. In addition, the rice root .aphidswere recoveredfrom a suctiontrap locatedin PayneCounty 12December 1965. Bird cherry oat aphids and greenbugswere common speciescollected throughout the state on wheat and barley. We can only speculatewhy the rice root aphid has becomeabundant the last few yearson wheat. Without magrification, it is difficult to distinguishbird cherry oat aphid from rice root aphid. It is possiblethat a mixtrue of the two specieswere tallied as the bird cherryoat aphid while samplingfor aphidsin wheatin previously publishedstudies. From ow experiencewith the rice root aphid, it appearsthat the aphid is more numerouswhen the temperatureis higher than normal and precipitationis lower than normal. Kieckhefer and Gustin (1967)came to similar conclusions. Theseconditions producedry, friable soil with numerousairspaces, and an environmentthat appearsideal for subtenanean aphids(Kieckhefer and Gustin 1967). However,we found that after heavyprecipitation there was no evidenceof a dramatic reduction in numbersof the rice root aphidsin wheat fields. For instance, on 30-31 January 2002, three inches of fteezing precipitation occuned in Garfield County, OK. It was recordedas the worst ice storm to ev"r occ* in Oklahoma. when the fields were sampledon I I February 2002, 23.l o/oof the aphids in the l0 fields were rice root aphids(Table t). Again in the fall of 2002,beforesamiling the field located in -PayneCounty, OK on 30 October2002, the field received3.5 inchesofirecipitation over a,2-3 day period. we recovered1,068 rice root aphidsfrom soil t".ibr taken on 30 october 2002, thtee days after-tle heavy rain (Table 2). This was the highestpopulation of live ri99 root aphids recoveredfrom the field during trtat rat sampling ipparently the ri'oioa. aphid-has the ability to survive for at leastseueral days when soil is l"*Oat"a *lth water. A reJate{ speciesof aphid called the waterlily aphid,irhopalosiphum nympthaeae (L.), cm survive for several days submergedin water, becausethe'aphid"pp**tf has specialized appendages which can Eap air bubblesbetween their legs Qraacnae-o wlo"t "rto use;. From our study and a review ofthe existing titerature,it appearsknowledge oithe biology and ecology of the rice root aphid and its=affectson *rtiut t;*h and yield are currently insufficient to accuratelygauge its importanceas an economic pest of wheat in Oklahoma. Additional study is neeOeOto deiermine the economic i-iott-.. of this potential pest aphid speciesin Oklatromawheat production ACKNOWLEDGMENT _ we are gratefur to Aaron Miller, Keith Mirkes, and Justin spurlin for technical with T:t.l*." this project. wade Frenchand Geraldwiide provioJ iJffi rerriewsortle initial version popham, of the manuscfpl Tom usDA-ARs'spA stutirti"ian,conducted the :t"tittitfl using SAS. Mention oit "a" namesor cornmercialproducis in this article rs solely Tlv.9. ,Ior the purpose of providing specific information and does not imply recommendationor endorsementby the u.s. Departrnentof Agriculture. LITERATTJRECITED Blackman,R. L., and v. !.- E-astop.r9g4. Aphids on the world's crops:an identification guide. Wiley, New york. chapiu J. w., J. s. Thomas,S. M. Gray,D. M. smith, and s. E. Halbert. 2001. Seasonal abundanceof aphids (Homoptera: Aphididae) in wheat, and their role as barley 251 yellow dwarf virus vectorsin the SouthCarolina Coastal Plain. J. Econ. Entomol. 94: 4t0-421. Doncaster,J. P. 1956. The rice root aphid. Bull. Entomol.Res. 42: 741-747. Gray, S. M., J. W. Chapin,D. M. Smith, N. Banerjee,and J. S. Thomas. 1998. Barley yellow dwarf luteovirusesand their predominantaphid vectorsin winter wheatgrown in SouthCarolina. Plant Dis. 82:1328-1333. Jedlinski,H. 1981. Rice root aphid,Rhopalosiphum ntfiabdorninalis, a vector of barley yellow dwarf virus in lllinois, andthe diseasecomplex. Plant Dis. 65 :975-978. Kieckhefer, R. W., and J. L. Gellner. 1992. Yield lossesin winter wheat causedby low- densitycereal aphid populations. Agron. J. 84:I 80-I 83. Kieckhefer, R. W., and R. D. Gustin. 1967. Cerealaphids in SouthDakota. I. Observations on autumnalbionomics. Ann. Entomol.Soc. Amer. 60: 514-516. MacRae,I. V., and N. N. Winchester.1988. A new host plant in B.C. for Rhopalosiphum nynphaeae(Homoptera: Aphididae). J. Entomol.Soc. Brit. Columbia.85:98. Paliwal,Y. C. 1980. Transmissionof barleyyellow dwarf virus isolatesby the cerealroot aphrdRhopalosiphum rufi.abdominalis. Can. J. Plant Pathol. 2: 90-92. Patch,E. M. 1938. Food-plantcatalogue of the aphidsof the world. MaineAgric. Exp. Stn. Bull.393:36431. Pike,K. S., L. Boydston,and D. Allison. 1990. Alate aphidviviparae associated with small grains in North America: A key and morphometric characterization. J. Kansas Entomol.Soc. 63: 559-602. Robinson,A. G., and S. J. Hsu. 1963. Host plant recordsand biology of aphidson cer€al grainsand grassesin Manitoba (Homoptera:Aphididae). Can. Entomol. 95: 13+-,137' Royer, T. A. 1999. Aphid numbersin wheat are variable. OklahomaState Crop Statement. 2;6-7. Se$ Institute. 1990. PROC user's manual, version 6th Ed' SAS Institute, Cary, North Carolina. Starks, K. J., and R. L. Burton. 1977. Greerbugs:Determinging biotlryes, culturing, and screening for plant resistance, with notes on rearing parasitioids. USDA-ARS TechnicalBulletin 1556.12pp. Tsai, J. H., and Y. H. Liu. 1998. Effect of temperatureon development,survivorship, and reproductionof rice root aphid (Homoptera:Aphididae). Environ. Entomol. 27:662- 666. True, R. R., F. M. Epplin, E. G. Krenzer,Jr., and G. W. Horn. 2001. A surueyof wheat production and wheat forage use practicesin Oklahoma. OklatromaAgric. Exp. Stt. Bull.B-815.34pp. Van Cleave,H. W. 1970. A preliminarylist of aphidand host plant records from Oklahoma (Homoptera).J. KansasEntomol. Soc. 43: 101-111. 252 vol.29 NO.4 SOUTIII'TESTERNENTOMOLOGIST DEC.2004 EFFECT OF PHAGOSTIMUT.AI{TSIN ARTIFICIAL DIETS ON HONEY BEE FEEDINGBEHAVIOR Anita Hannaand Justin O. Schmidt Carl HaydenBee ResearchCenter, ARS, USDA, 2000 E. Allen Rd., T\rcson, AZ85719 ABSTRACT The role of phagostimulantsand diet fomrulation in dietary choices made by honey bees,lprs mellifera L., was studied in laboratory. Diets in which sucroseor -andpure glucosl comprised the added carbohydratesweetener rapidly lost water, hardened, becacre difficult for the beesto eat. Reductionof the glucoselevel to 75Yoor less and replacement of sucrosewith fructoseyielded a soft, acceptablediet texture that lost little water and could be readily consumed by bees. A sugar combination of 50o/ofructose and 50%oglucose "pj9.ud ideal for incorporation in bee diets. Feeding choice preferencetests in tI: days old honeybees revealed_thatadlition of phagostimulantinsrease.d diet consumptionover unEeatedconhols. Addition of as little as l-5o/opollen exhact sigrificantll enhanced dietary feeding behavior in all diets-tested. Phagostimulant eni-ancementof seven commercialhoney bee substitutediets or dietary ingredientswas analped for nutritional potential in longevity tests. The diets diff€red inthe time lengthsofbee survivA, suggesting pot€ntial differencesamong the nu&itional values of the aiec. A complicating Aitor was the p-resenceof heavy infeslationsof varroa mites, Yarroa destnraor Aidqg6;nb true6aru 9n $e !ees. Phagostimulantsderived from pollen appearto play an important role in fecding behavior of honey bees and the potential for dJvefoping more successfulartificial diets for bees. INTRODUCTION - Honey bee foragersare lnown to collect a wide variety of nutritive and non-nutitive substancesthat are presumedto be food by the foragers(Dietz lgTS). The generalview had been that bees were not discriminatory in food setectionand based deci-sionsmainly on texture or particle size of the pollen or powdery substancecollected. A role of tastewas not recognized. The mYth that beeswere not selectivein their food preferencewas challenged with experimentsthat showed foragers given choices of pollen from four different piant speciesexhibited distinct pre_ferencesarnong the pollen types (Schmidt 19g2). Likeudse, young ntrrse bees that actually cons-umethe pollen brought to the hive e*trititeO snotrj gry_feren99sfor pollen of some speciesover those'of othi species(Schmidt and Johnson 1984). -Nurse bees given a choice of pollen ftom individualspecies, ve$us a mixtwe of pollen from those sarnespecies, preferred the mixed pollen (Schmidt i984). Thesereports indicated that bees likely. use- taste, particularly the presence of phagostimulants, iolle,n-produced ia their choiceof food sollrces,a postulationconfirmed *it f"o6log ni"t. involving extractsfrom polle,nadded to a laboratoryartificial dia (schmidt l9g5). Diets that substituteother proteins fol nollen are an important aspectof modemhoney bee management.Substitute diets are administeredwhen beesare weakeneddue to periods 253 ofpollen dearth,population loss after pesticide sprayrng,or from heavy varoa or bacheal, Acarapis woodi @ewie),. mite infestation rH"yart 1970, Standifer et al. l97g). Additionally, substitutediet is-neededto manipulatecolonies to'have optimal populationsat the timesof nectarflows and f9r crop pollinati,on(Shuel and Dixon l9ti6, feng et al. l9g4). Currently, all available substitute diets lack polien phagostinulants, have ingredient or texture problems,and are generallypoorly acceptedby the bees. An effective diet must provide most of the nutritive elements for growth and developmentofhoney bees,have overall higfr nutritional value, have a proper texhse and consistency,and not be toxic (Schmidt 1984). Crucial elementsin insect diets, including those of honey bees, are the presenceof phagostimulantsand the absenceof feedin! detenents(Schmidt 1985), The effectiveness of nutritive diets can be evaluated both behaviorally and physiologically(Schmidt et al. 1987).A behavioralmeasure is relativequantities of diets consumedin choice tests. An estimateof the physiologicalvalue of a diet is the longevity of animals fed the diet. In order to improve the atbactivenessof artificial diets to honey bees and to increaseoptimal feeding,we testedthe role of phagostimulantsin dietary acc€ptance and nutritional value in bees. The objectives of these studies were: 1) to determinethe palatability of diets for honey bees(Texture and Moisture Test), 2) to determinethe role of phagostimulantin diet choicesofhoney bees(Feeding Preferences), and 3) to determinethe nutitional value of diets for honey bees(Survival Studies). MATERIALS ANDMETIIODS Honey bee corbicular pollen from mixed floral sources was obtained from colonies locatednear Siena Vista" CochiseCounty, Arizon4 during the spring 2001. The pollen was storedat -20'C until exhacted. Pollen was extractedfor 8 h in a soxhlet extactor with ethyl acetate@urdick and Jackson,Muskegoq MI), and the liquid fraction was evaporatedto near drlmess,dissolved in acetone,and stored at -2OoCuntil used. Extract arnountused in the testsis listed as a percentage.For example,100 g ofa diet containing 5% pollen extact would contain the extract of 5 g of pollen addedto 100 g dry weight of diet. Test diets were obtainedfrom commercialsources and give,nthe following abbreviated names: Bee Pol (International Ingredients, St. Louis, MO); BeePro (Mann Lake, Hackensack, MN); Beltsville bee diet, or BBB @io-Serv, Frenchtown, NJ); Casein (IntemationalIngredients); Dadant @rood Builderru, Dadant& Sons,Hamilton, IL); Kelley ('?ollen Substitute",Walter T. Kelley, Clarkson,KT); and Rice Protein (Product STD 50- MF1000, Intemational Ingredients). Phagostimulantwas addedinto diets by pipetting the appropriateamount of acetonesolution into a beaker,evaporating solvent just to dryness, and mixing dry diet with the extact until uniformity was achieved. At this point, enough distilled water was addedand mixed to form a moist cake. When not fed to bees,test diets were kept at 4oCfor up to one week, All testswere conductedin an environmentalroom designedto simulatethe conditions inside the brood chamberof a normal honey bee hive. The room was illuminated by red fluorescentlights and maintainedat34oC nd60-70% relative humidity. Textureand Moisture Test. Test diets in hives or in the environmentalroom often becomehard, dry, and difficult for beesto consume. Preliminary tests revealedthat diets formulated with sucrosequickly becamehard and rmsuitable. Since water loss within 24 hours from diets appearedto be good measureofpotential hardnessand unpalatability to bees, we determinedwater loss from diets formulated with different ratios of the sugars glucose and fructose. Five diet treafinentsconsisting of 50%ototal sugar were formulated with the following ratios of fructosoand glucose:100:0; 75:25; 50:50;25:75;0:100. The test diets were placed in 50 mm Petri dishesin the environmentalroom and weigbed daily for water gain or loss. 254 Feeding Preferences. Sevencommercial diets or dietary ingredientswere testedwith five phagostimulanttreatnents: exhact from pollen equivalentto l\o,2.syo, syo, lOyoNd 20%'pol'ien added to the diet. All diets were made by adding equal amormtsof 50:50 fructose:glucoseto the dry diets and mixing with enough distilled water to make a soft kneadabletexture. For the tests,about t25 (15 g) 1- to 3-dayold workerbees were placed in 9x6xt5 cm acrylic plastic and screencages as describedby Schmidt(1984) and Schmidtet al' (1995). Each test consistedofthree replications. The beeswere offered a choiceoftest diet plus phagostimulantor test diet alone. They were also provided water, a piece of beeswax foundation upon which to rest, and a 50% sucrosesolution. The test and referencediets in eachcage were weighed after 48 hours, replacedwith fiesh diet in reversedcage positions, and weighed again after 48 hours. The beeswere then releasedto a hive. A t-test was used to show the differe,ncebetween test diets and control (SAS Institute 1994)with a sigrificant valueof P <0.05. Suntival Studies. The effective,nessof the phagostimulantenhanced diets on the survival and longevity ofbees was conductedon eachofthe sevendiets. Testsfollowed the proceduresofSchmidt et al. (1987,1995) and consisted ofthree replicateseach. Testswere conductedin the environmentalroom using 60 l-day old bees,heavily infested with Tarroa destructor mites, placed in acrylic plastic and screencages (9x6xl5 cm). Each cage was provided water, a piece of beeswax foundation for the bees to rest uporl 50olosucrose solution, and a single test diet. Diet consumptionwas measued evety 2 or 3 days until the last bee died. Mortality curves were generatedwith survival analysisby SAS Proc (SAS Institute 1994) followed by Wilcoxon and Iog-Rank tests;diet consumptionanalysis was by analysisofvariance. Ratesof Diet Consutnption.The comparisonof diet consumptionof honey bees fed several artificial diets were conducted as described by Schmidt et al. (1995). The experimentalconditions were describedin the section on survival studies. The diets were replaced every second or third day for 30 days and data were anallzed by analysis of variance(SPSS 1990). RESIJLTSAND DISCUSSION Tetctureand Moisture Test kritial treafrnentsin which sucrosewas used as the sugar tended to lose water rapidly, becomevery hard within 24 hours, and were difficult for the bees to consume. After dietary sugarcwer€ changedto 50%ofructose and,50o/o glucose, evaporationrates decreased dramatically: Bee Pol from25.8Yoto 4.8vo:Beepro from37.6% to l2.9To;Dadant from29.9%oto ll.6o/o;Kelley from 38.3%tol2.8%(Table l). Diets consistingof glucoseas the only addedzugar lost considerableweight over a 48 how period andbecame hard. In conhast,diets containingat least2l%o fructoseeither lost TABLE 1. Mean P€rcentageWater loss from Test Diets Formulated Using Sucroseor 50:50Fructose:Glucose as the SugarComponent. Diets Sucrose(Mean + SD) 50:50Fructose:Glucose FrobaUilitl BeePro 37.58 + 294 12.86+1.54 0.0001 CaseinProtein 7.85+ 0.94 6.02* 0.65 N.S Dadant 29.90+2.31 tt.62+2.34 0.0001 Kelley 38.33+ 4.53 12.77+ 1.99 0.0001 Rice Protein 36.22+3.94 4.26+0.76 0.0001 "Difference in water loss betweensucrose and 50:50 fructose:glucose(t-test) 255 {-r !.2 a :'l-3 E !-s ia Frucaos 100 73 50 23 0 Cl!@ 0 ,3 130 73 l|X) Sugrr rrtlor (%) FIG. 1. Water Loss from Bee Diets Made with Fructoseand Glucose. liltle yater or gainedsome afinospheric water to increaseslightly in weight (Fig. l). Those digts that gainedweight were softer after 48 hows than at th; b;giDning ortfu test. For the other tests, we chosethe compositionof 50% fructoseand 50% glucoJ" to b" addedto the diets, in part becausethis ratio maintained a very good diet textwe and in part because holef-' the normal sugar sourcein the hive, is composedof about equal portions of fructose and glucose-(whitq 1992). Experimentsusing sucroseand high fructose com qmry were not successfulbecause these sugars tended to allow large water lossesfrom the diets laata not shown). FeedingPreferences. Honey beesreadily discriminatedbetween artificial diets with or without phagostimulantextact (Fig. 2). For many diets the difference was evident even with addition of as little as l% @eltsville Bee Diet, Caseinprotein, Rice Protein) or 2.5Yo pollen extact @ee Pol, Dadant, Kelley). only the BeePro diet required extact of 5% pollen to caus€a difference. In general,increases in phagostimulantlevels abovethoSe first eliciting preferenceproduced little increasein prefercnce. This effect suggeststhat honey bee feeding behavior might be stongly influenced by a threshold effect; that is, once a thresholdphagostimulant concentration is reachedto signal that tlrc food is recognizedand acceptable,little added stimulus enhancementoccurs. In practical terms this indicatesthat very little pollen extact, typically less than 5olo, is required to effect meaningful improvementof the acceptanceof the substitutediet by the bees. This, of course,zursumes all other factors in the diet are suitablefor the bees. Surttival Studies. Cumulative survival curves of honey bees fed different diets are shown in Fig. 3. The averagelife spanofthe beesfed various diets rangedfrom 7 to 12.5 days, with a me€urof 8.9 days (Fig. 4). sigrrificant differencesamong groups of diets were observed (P <0.0001, wilcoxon and Log-Rank Tests). Three of the commercial diets, Kelley, Bee Pol and BeePro,prolonged the life spanofhoney beesrelative to the average. Rotesof Diet Constrmption.Diet consumptionrates from sevendiets are showuin Fig. 5. The bees generally consumeddiet most rapidly during the fint 5 days. Thereafter, consumption decreasedsharply and by day l0 had almost ceased. Analysis of variance shows that the mean rate of consumptionhad a highly sigrificant effect on the total of consumptionof the diets (P<0.05). 256 n F l...... _ x fa 'mmot ,+ 6' *---ffiIlutrtrI x"E iF => tN SE: E: N dj o!) 2 \rt O ,ru -E E& ! kl q) ,- o.9 6E gtr tW o E$ ts *-ffi s S Eil q999 H Eg FEmroc ouc *[m E8 ES 'lmruuuruilI i'tvE Eii F! -C('$€ 'N e l ao -N xi 3s t E ':c.'l :! frr $d Xg '-ffi o tri: C) { ':E d -ffi I LrE x - m.=Hg oboo '-flm d;;d ;F poonnoc @uc 9,a *-flIIIIIIItr oc) E t;{ *N 8i x- T :sAO N 6l .:-+E ct , nE E€ .t *--m EE g* 'ffi -fi?d .q r-ffi s EE VHUJs [|qa IF o w= lputr|muuc r!pr 251 Ba -H : o 8 -tF? - 5 f F '{IITTTTITE8 ;" ?g t ooc -{Iil]ItrTilililI s€ E€Eg a E-k nE vc 3E €? o g -c(, " i5'g SE E -'Q E E E9 ,%. u!I "i C s9 iR ,- fr-6 $d ;d FiG EN -w6 (J e €- - g.q s + !l .^ q9q9q lqacac E6 oo sO pun$o9r|lu9 uqc ECa FerlnNoJ i-d FN u^o E8 do !i oii '9 t'' €c '-F 6 3 ?E .. (:tr x e; ox *- ^ .ztq '{mm SE sg 2E a x9 'flIII]TMTIIITI-IT eE E€ E TF -o Yaii o o g -(, :E F dr dE *H e E 5s ,-%, uuJ of E 6 K s! "sE 60 v-- c.i c $E '-w 5 a6b =0 s *--ffi s E 5E 1E eqqa caqq ?o 9fi ir rtl paun3uoa auso peunauot 3use 258 0, BeePol BeePro 0. 0. 0. 2 4 6 8 10121416182022242628 2 4 6 8 101214161820?2242628 Age (Days) 1. .- -- 't, i t.h-- ^^l BBD Dadant 24618 10 12 14 16 18 20 22 24 26 28 2 4 6 8 10121416182022242528 Age (Da)rs) 1. I 0. 0. Casein Protein Rice Protein 0. 0. 0, 0. 0. 0. i 2 4 6 8 1012141618202224m28 2 4 6 A 10 12 14 16 182022,242I52tt Age (Days) Age (Days) l _.,,1 Kelley FIG 3. Survival Profiles of Honey Bees Fed Single SourceDiets. w.qtl t-l 0.11 | L- v.m:^^l | j 2 4 6 8'101214161820222426281 !_t i As€(Days) i L"..,"--"*.-,.*.*,.,.*"."* *..".---.,.-,..-..,."-.,."-,.,,",",,.,,",i 259 I a a a a a '<€F'-' r <€rr.g*:- al ' I "gF* ffi'. I a - I a a FIG. 4. Mean Longevitiesof Honey BeesFed Single SourceDiets. Diamond $hapeis Group Mean of Each Diet. 18 1A Kcllcy 14 o BccPro E 10 U CescinP rot.in c L Ricr Pr6tcin 4 Dadant BBD '10 0 2 4 6 I 12 14 16 18 20 22 a6 Dry of Feeding FIG. 5. CumulativeFood Consumptionof Honey BeesFed Single SourceDiets. Schmidt et al. (1987) reported a similar study using different pollens instead of substitutediets. ln that study beeswere fed 25 different pure pollens plus severalblends of pollen as sole food sources(other than sugar). Survival times for thesebees (30-35 days) 260 l were better than in the presentstudy (8.9 days). In another study, Schmidt et al. (1995) anallzed tbree tlpes ofpollen (sunflower,sesarne and rape) and obtainedaverage life spans of 31, 33, and 5l days,respectively. The differencebetween our studiesand the earlier studiesare both diet types and parasiteinfections of the bees. The first two shrdiesfed bees with pollen, their natural food source;we fed pollen-freeartificial diets enhancedwith minor quantitiesof pollen flavor. Pollen provides all the nutrientsneeded by beesfor growth and development,except for energy, and is readily digested (Schmidt and Buchmann 1985, 1992). Becausethe diets testedin the current experimentslikely do not effectively replace the nutrients in pollen and becausethe beesused were heavily infested with varoa mites, resultsfrom this study cannotbe directly comparedto thoseofthe earlier studies. ACKNOWLEDGMENT We are indebted to Leonard Hines for providing pollen and Steve Thoenes and Hayward Spanglerfor manuscriptreviews. LITERATTIRE CITED Dietz, A. 1975.Nutrition of the adulthoneybee, pp. 125-156In Dadantand Sons [eds.], The Hive and The Honey Bee. Dadantand Sons,Hamilton, IL. Haydak,M. H. 1970. Honeybeenutrition. Ann. Rev.Entomol. 15: 143-156. Peng,Y. S., Marston,J. M., and O. Kaftanoglu.1984. Effect of supplementalfeeding of honeybee(Hymenoptera: Apidae) populations and the economicvalue of supplemental feedingfor productionof package-bees.J. Econ.Entomol: 7 7 : 632-636. SAS Institute1994. SAS/STATUser's Guide. SASInstitute Inc.,4s edition. Schmidt,J. O. 1982.Pollen foragingpreferences of honey bees.Southwest. Entomol. 7: 255-259. Schmidt, J. O. 1984. Feeding preferencesof Apis mellifera L. (Hymenoptera:Apidae): individualversus mixed pollen species. J. KansasEntomol. Soc. 57: 323-327. Schmidt,J. O. 1985. Phagostimulantsin pollen. J. Apic. Res.24: 107-114. Schmidt,J. O., and S. L. Buchmann.1985. Pollen digestion and nitrogen utilization by Apis melliferaL. (Hymenoptera:Apidae). Comp. Biochern. Physiol. 82A:499-503. Schmidt,J. O., and S. L. Buchmann.1992. Otherproducts of the hive, pp. 927-988.In J. Graham[ed.], The Hive and The Honey Bee. Dadantand Sons,Hamiltoo, IL. Schmidt, J. O., and B. E. Jobnson. 1984. Pollen feeding preferenceof Apis mellifera, a polylecticbee. Southwest. Entomol. 9: 4l-47. Schmidt,J. O., S. C. Thoenes,and M. D. Levin. 1987. Survival of honey bees,Apis mellifera (Hymenoptera:Apidae), fed variouspollen sources. Ann. Entomol. Soc. Amer.80:176-183. Schmidt,L. S., J. O. Schmidt,H. Rao,W. Wang,and L. Xu. 1995. Feedingpreference and survival of young worker honey bees (Hmenoptera:Apidae) fed rape, sesame,and sunflowerpollen. J. Econ.Entomol. 88: l59l-1595. Shuel,R. W., and S. E. Dixon. 1986. An artificial diet for laboratoryrearing of honeybees. J.Apic. Res.25: 35-43. SPSSInstitute 1990. SPSSBase System User's Guide. Norusis/SpSSLrc.pp. 2ll-215. Standifer,L. N., F. E. Moeller, N. M. Kauffeld, E. W. Herbert,and H. Shimanuki.1978. Suppleme'ntalfeeding of honeybee colonies. USDA InformationBulletin No. 413. 8 pps. White, J. W. Jr. 1992. Honey, pp. 869-925./z J. Graham [Ed.], The Hive and The Honey Bee. Dadantand Sons,Hamilton, IL. 261 vol.29 NO.4 SOUTIIWESTERNENTOMOLOGIST DEC.2004 TRANSGENIC.BFCORN AFFECTS SUGARCANE BORERI IN LOUISIANA C. D. McAlli$#, K. P. Bischof, K. A. Gravois3,H. P. Schexnaydera,Jr., andT. E. Reagan2 ABSTRACT Only one larva of the sugarcanebora, Diatraea saccharalis (F.), from 360 newly emerged larvae feeding on Bacillus thuringiensis tansgenic com @ioneer 34R06) in a replicatedfield experimentduring 1998,survived to pupation. Survivalwas high (94lawae) in all plots of the non-transformedhybrid variety (Pioneer 3489). The CryIA(b) gene transformed from Monsanto's YieldGard@ technology proved to be highly effective in suppressingartificial and natural infestationsof the sugarcaneborer in com. In 1999, the hansformedhybrid variety MON8IO x MON840 had the lowest percentagesugarcane borer infestationcompared to other rt hybrid varietiesand the non-8t hybrid variety. Theseresults indicate that .Bf-corn may have a potential role as a component in the sugarcaneborer managementsystem when com might be produced during fallow periods of sugarcane production in southem Louisiana. 8t-com may be planted to help minimize the usual increaseofthe sugarcaneborer whencom senesces,However, depending on the padicular transgenicvariety selected,these data show that there can be a substanstialdifference in the proportion of larval mortality and the potential amountof selectionpressure exerted against the sugarcaneborer, INTRODUCTION The sugarcaneborer, Diatraea saccharalis (F.) (Lepidoptera:Crambidae), is often a serious pest of com, Zea mays L., in the sugarcaneagroecosystem in southern louisiana (Flpn et al. 1984). Substantialyield loss is due to tunneling in the baseof the ear, shank, and lower intemodes. Infestationsbeginning at or before silking causethe greatestimpact on yield, reducingprimary earweight (Flynnand Reagan 1984). The com cultivar Funk's 581 has been shown to causea 3-fold and l.7-fold increasein D. saccharalis pupal production comparedto commerciallygrown sugarcanecultivan CP65-357and CP6l-37, respectively. The increasewas attributedto the low abundanceofpredatoryarthropods (Reagan and Flynn 1986). In anotherstudy, fecundity was much greaterin D. saccharalisproduced on com than on other gramineoushosts (Bessin and Reagan1990). Previous sugarcane field studies (Wier et al. 1989; Schexnayder et al. 1992; Rodriguezet al. 1994,1995)have assessedvarious formulations of Bacillus thuringiensis var. hnstaki for D. saccharclis conEol at rates ranging from 5.6 ta 12.0 BIU's (billion international units). Treatmentswith their levels of control were Bactec Beman BT III (32Yo), Condor Q7o/o),and Javelin (26 and 43%Q. All .Bt treafinents were significantly inferior to commercialinsecticides. A seriesof laboratorydosage-mortality bioassays using several formulations of .Br were conductedon the soybeanloopa, Pseudoplusiaincludens L€pidoptera'Crambidse 'DepartnentI ofEnlomology, LouisianaStale Agrioultural Center, 402 Life SciencesBldg., BatonRouge, LA 70803. ' Sugr ResearchStation, Louisiana Agricultnral ExperimentStation, Louisiana State Agicultural Center,5755 LSU Ag Ro8d,st. Gabriel,I^A,70776. a louisiana DepartmentofAgriculture andForestry, Baton Rouge, LA 70806. 263 (Walker).Mascarenhas et al. (1998) reportedthat field collectedP. includenswere more resistantthan a susceptiblelaboratory colony, Field populations of P. includens collected from soybeans,Glycine max (L) Merill, and Br-cotton crylA(c), Gossypiumhirsutum L., showeda reducedsusceptibility to CondorXL comparedto a laboratory-rearedsusceptible strain. The limited persistenceof 8t insecticides a major factor slowing the buildup of resistance.Refuges are the best way to slow the developmentof resistanceto Bt in areas with transgeniccrops (Tabashnik 1994). Field and laboratorystudies with the cryIAO) geneof Br transgeniccom haveshown suppressionof D. grandiosellaDyar(Williams et al. 1997,1998). A reportfrom Cubaof Bl transformed sugarcane also has indicated a potential suppression of D. saccharalis (Arencibia et al. 1997). South African sugarcanestudies with the Eldana saccharina stalk borer have shown suppressionwith the CryIA(c) cloned gene infoduced into an isolate of Pseudomonasfluorescens (Herrera et al. 1994). Becauseof the potential for Bf tansgenic tecbnologyto be integratedinto an area-wideD, saccharalismanagement system, a two-year study was designedto assessD. saccharalisfield susceptibilityto 8r-com with the Cry IAO) genein severalcom cultivars. MATERIALS AND METHODS Com in eight row plots (rows on 0.9 m centers),1l m long (0.008ha) witb 1.2 m alleyswas plantedon a Commercesilty clay loam soil 4 June 1998at the LouisianaState Univenity Agricultural Centerbranch experimentstation at St. Gabriel. Pioneer3489 (non- Bt) and Pioneer 34R06 (transformedvariety with MonsantoYieldGard@ technology - CryIA(b) gene)were planted. A four-row planter was calibratedto plant seedsspaced 0.1 to 0.15 m with plots arrangedin six replicationsof a randomizedcomplete block design. No insecticideswere usedin the soil or on the foliage during the study.Due to unusuallylow naoral infestationsof D. saccharalis (early season),20 corn plants were randomly selected from the center four rows for artificial infestation on 29 July (during early ear formation, prior to tassel). Each plant was infested with three newly-hatchedlarvae obtained from a Louisiana State University Agricultural Center laboratory colony that originated from a colonycollected eight monthspreviously by Dr. W. H. White of the USDA-ARS Sugarcane ResearchUnit in Houma, La. To expediteinfestation, the larvae were placed three per vial into O.I-dramplastic centrifuge tubes in the laboratorybefore being taken to the field. Com plantswere sampled for D. saccharalislarvae on 13 (blisterstage) and 25 Augustand l0 and 16 September(dent stage), using a destructivesampling technique with carefuldissection of all parts of the entire stalk and ears. This samplingtechnique consisted of removing each leafand leafsheath from the stalk and visually inspectingand cutting up the stalk, ears,and leaf sheath and searchingfor live and dead D. saccharalis larvae. Six replications were sampled13 August, other plants from four of the six replicationswere sampled25 August, with the remaining two replications sampled on l0 Septemberdue to sudden unsuitable weatherconditions preventing sample completion on 25 August. Samplingon 16 September (five plants per plot) was conductedto assesslate seasonnatural infestationson plants away from thosethat had beenartificially infested. All larvaecollected from plots in this study were takento the laboratory,placed on artificial diet (modification of Hensleyand Hammond 1968),and allowedto feed andpupate. Pupaewere then weighed. The meannumber of D. saccharalislarvae collected in 1998was anallzedusing an unpairedtwo-tailed Student's r- test(SAS Institute 1999). In 1999,com wasplanted to eigfit-rowplots (rowson 1.9m centers),9.8 m long and 0.9 m alleys, on a Commirce silty clay loam soil 19 May at the t ouisiana StateUniversity Agriculturil Centerbranch experimentstation at St. Gabriel. The treatmentsused were B73 * vtotz (non-Bt com), MON8IO x MON840,B70 x MoN840, and MoN8lo x M017 (the 264 last three treatnents were Bt com transformedwith Monsanto's YieldGuard@technology) arrangedin a randomizedcomplete block design with six replications. The sameartificial infestation techniquewas used in the secondyear that was used in the 1998 study prior to tasseland was conductedon 6 July 1999. Eighteencorn plantsper plot weresample-d on 23 h'ly at the silk stageand twelve stalks per plot were sampledon t f nogust (milli stage)for D. saccharalislarvae using the destructivesampling technique describedfor ihe l99g-study. D. saccharalis infestationsand mean larvaeper plant were analyzed,usingpRoC GLM and means were separatedusing Ryan-Eimot-Gabriel-welsch(REGWe) Multiple Range Test (-SAS krstitute1999). Due to the increasednumber of treafinentsin ile secondy.* rtrdy, a differentstatistical analysis was used. RESULTSAND DISCUSSION Of the 360 first-instar larvae artificially infested (20 plants x tlree per plant x six replications),only one survivedfrom the totalBt infestationduring r99s G;ble i). on the 13 Augustsampling date (15 daysafter infestation), 45 D. sacchaiaftblarvae were collected the !91 ryn-Bl plots and were transferredto aftifi;ial diet with an urr".g" pupal weight of 0.15 g (sD = = 0.044,SE 0.008);whereas, two D. sacchararisrarvae (only one survivedto pupation, with a weight of 0.09 g) were obtainedfrom the samenumbeiof sampledBr plants thlt_rye1e infested previously. Later sampling (25 August and l0 sepiemti.y from both arfificial and natural infestationsrevealed no larvae in ttre rl plots. Forty-five, lt, "nd ta live D. saccharalis were collected in non-gr plots on 13 August, zi eug*t, and l0 s9n^t9m!er'respectively. Destructivesampling oinaturally infestei plants revealedthat five of 30 plants (non-.Br) containedlive larvae,wf,"."ar none of the 30 desfructivelysampled gt plantswere infested.In 1999,MoN810 x MoN840 hadthe lo*.rt p"i."trtrg e D. saccharalis infestation (0.83%), the lowest mean number of larvae per plant (b.09) ad the lowest total number of D. saccharar,s per Jarvae 12! rotal planb diisecied rrbl rraure 2). The non.rgr corn Eeatmenthad the highestpercent D. saccharalis infestation 1io.auq, the highest mean number of D. saccharalu per plant (4.3), and the highest total number ofsugarcane borer 120 falae {or total plants dissected(36). All .Br com varietieshad lower D. saccharalis infestation. Resultsfrom this studyindicate that transgenicBt commay have a potential place as an alternative crop grown during te spring/suriner fallow period in the-srrga."uneborer managementsystem in southemLouisiana. Additionally, utilization or tne ar gene in sugarcaneplants for managementof D. saccharalis on an area-wide basis could be considered of supplemental value to the sugarcanefarmers of southem Louisiana in divetsifting their managementsystem. Howevir, the survival rates on some of the Bt com cult-i-varsin the secondyear study are rather high, suggestingthat resistancecould becomea soon, {ob.fm especiallyin the absenceof atteition to resistancemanagement. of particular significancefor possible resistancemanagement, in comparisonto other field cropswith gl use,is that additional 8/ selectionpressure would be reduceddue to the absenceofany useof 'Br arthropod sprays. None of the currently labeled D. saccharalis insecticidal chemistries (diacylhydrazine,pyretbroid, organophosphate,carbamate) are known to exhibit a.Bt cross- resistancespectrum and Bl spraysare notlffeciive for use on sugarcane.With further study, the possibility exists that Bt com may be utilized as a trap c.op in the sugarcanesystem in Louisiana, by athacting D. saccharalis.allowing them to fee4 ano to oie Jn ae corn plants. However, at this poin! the relative atnactiveiess of com ior D. saccharalls oviposition remainsundetermined. 265 I IB o o (t LI ae o I i(ol E h q 6rl ool 6. ol E h0 {) e lES Eso a I I€E gE€ .3 I v I .s ql ()o NI U I i=lEcEE€E E I *l €EA 6l IEE; l€5 f E'?€._ l="soE s Fu vElo*l egl 6 s&l IES:€t*s e ';t EE€E ,!t c) Ic:B at-- k d ztg -t o\ lc) o\ s. rf, lA @ X l,o Fq5;e z o 6 xt- Cq fftExe a lst* Ff:P:"^ o I vl6l €l lsiE lgi€gI sll q o' l le..E€i?'sres F{ a () n ,e I rrl sl j sE IFEE F€gFgFR :l ol Fl EI .Fl s I 33 l;i F;s Figii 266 5c 3. q) € a ,il o a F tr sl BE F q) e &ls o qiv=o d0 3q o ) gl* a U) F ci 6a a tr{t A >*l CI t.' 3fi .E ({F "l N .k ES I h .G I gc ,s qc) !.1 ,-l tk t() €$ P;.E" ctl z € F I s Et€€ cr! l* E &13o oo tc E? B x 3, to gd *l I>r€ *F n z = tro €& ; IE I E_ F G' "-l Fi Sd-,E 0 ls <.g t4 l(! .tr € € g G { E X sE: c cflqx\l l,Eo tc E tc)€ zil=l tJ1 2 l€ tc) at oi € t6 Pi|l IEd ne tl l\tst iflIE -s € z1 tZ>.ogETE () (\ N 90 F- F- €ii hE ae E A\ !i b3 b s'iEZ; ho,4:,H,EdTE x >{ j E.E il:ls (r) H Eg E.clIr\ .:Ed FRI z'l,l I al*.8 F T d> E,Es{ ula g.o E'5 > qXa< sE€E fiI -o Fl I u) 8 .E,A.E sl 6 F Zt c! e'tr I 4 -79I #E€E dr5 s (E C! -ol"- € \ a'- -.: X .9'6 <'Y XQ z E:=^oE aS Bs ..i fi glE,=co = 9.e 6Z S*EE x ;FFx 5fl zo t?l-:lx tri .=l (tl t-ixx Elii9> F 267 ACKNOWLEDGMENT Appreciation is expressed to the late Ed Ostheimer, former graduate student, forassistancewith field work and to Dr. W. H. White of the USDA-ARS SugarcaneUnit at Houma for assistancewith the sugarcaneborer laboratory colony. Appreciation is extended to ProfessorsJeff Hoy and Seth Johnson,and ResearchAssociate Waseem Akbar (LSU AgCenter) for critical reviews of the manuscript. This manuscript is approved for publicationas no. 03-26-1322by the Director of the LouisianaAgricultural Experiment Station. LITERATI.JRECITED Arencibia,A., R. I. Y6zqtez,D. Prieto,P. Tdllez,E. R. Carmon4A. Coego,L. Hem6ndez, G. A. De la Riva, andG. Selman-Housein.1997. Transgenicsugarcane plants resistant to stemborer attack. Molec. Breed.3: 247-255. Bessin,R. T., and T. E. Reagan.1990. Fecundityof the sugarcaneborer (Lepidoptera: Pyralidae), as affected by larval developmenton gramineoushost plants. Environ. Entomol.l9: 635-639. Flynn, J. L., and T. E. Reagan. 1984. Corn phenologyin relation to natural and simulated infestationsof the sugarcaneborer (Lepidoptera:Pyralidae). J. Econ. Entomol' 77: t524-1529. Flynn, J. L., T. E. Reagan,and E. O. Ogunwolu. 1984. Establishmentand damageof the Sugarcaneborer (Lepidoptera:Pyralidae) in corn as influenced by plant development. J.Econ. Entomol. 77:691-697. Hensley,S.D. and A. M. Hammond,Jr. 1968. Laboratorytechniques for rearing the sugarcaneborer on artificial diet. J. Econ.Entomol' 6l: 1742'1743. Herrer4 G., S. J. Sn;rman,and J. A. Thomson. 1994. Constructionof a bioinsecticidalstrain of Pseudomonasfluorescens active against the sugarcaneborcr, Eldana sacchaina, Appl. Environ.Microbiol. 60: 682-690. Mascarenhas,R. N., D.J. Boethel,B. R. Leonard,M. L. Boyd, and G. C' Clemens. 1998. Resistance monitoring to Bacillus thuringiensis insecticides for soybean loopers (Lepidoptera:Noctuidae) collected from soybeanand hansgenicBt-+otton. J. Econ. Entomol.9l : 1044-1050. Reagan,T. E. andJ. L. Flynn. 1986. Insectpest management of sweetsorghum in sugarcane production systemsof Iouisiana: problerns and integration, pp. 227-239. 1n W. H. Smith [ed.] BiomassEnergy Development' Plenum, New York' Rodriguez,L. M., E. A. Ostheimer,and T. E. Reagan. 1994. Small plot insecticidetrials, 1993. ArthropodManagement Tests 19: 278(142F). Rodriguez,L. M., E. Ostheimer,A. Woolwine,and T. Reagan.1995. Smallplot insecticide trial againstthe sugarcaneborer, 1994. ArthropodManagement Tests 20: 256 (133F). SASkrstitute. 1999. SAS InstituteInc., SAS@Procedures Guide, Version 8, Cary,NC, 1643 pp. Schexnayder,H. P., C. A. White, and T. E. Reagan. 1992. Sugarcaneborer control, 1991. lnsecticide& AcaricideTests l7: 287(129F). Tabasnik,B. E. 1994. Evolution of resistanceto Bacillus thruringiensis. Ann. Rev. Entomol. 39:.47-79. Wier, A. T., R. T. Bessin,T. E. Reagan,and E. J. Leblanc,Jr. 1989. Sugarcaneborer control, small plot insecticidetrial, 1988. Insecticide& Acaricide Tests 14: 285 (152F). 268 l Williams, W. P., J. B. Sagers,J. A. Hanten,F. M. Davis, and P. M. Buckley. 1997. Transgenic com evaluated for resistanceto fall annywonn and southwestemcom borer. Crop Sci. 37: 957-962. Williams, W. P., P. M. Buckley, J. B. Sagers,and J. A. Hanten. 1998. Evaluationof fransgenic com for resistance to corn earworm (Lepidoptera: Noctuidae), fall arm) tronn (Lepidoptera: Noctuidae), and souttrwesterncorn borer (Lepidoptera: Crambidae)in a laboratorybioassay. J. Agric. Entomol.l5: 105-112. 269 L- vol.29 NO.4 SOUTTIVTESTERNENTOMOLOGIST DEC.2004 THE MICROSPORIDITIM THELOHANIASOLENOPSAE IN RED IMPORTED FIRE ANTS(HYMENoPTERA:FoRMICIDAE)FRoMLoUISIANAPASTURES Y. Y. Sokolova,R. L. Bossard,J. R. Fuxa,D.W' Sanson,and L' D' Foil Departmentof Entomology,Louisiana State University AgCenter,Baton RougeLA 70803 ABSTRACT Between25 March and 15 May 2002, red imported fire ants,Solenopsis invictae Buren,were collected from five pasturessubdivided into 20 quadrantsat the Rosepine ResearchStation in Rosepine,Louisiana. Red imported fire ants (RIFA) collectedfrom 4 quadrantswere positivl for a microsporidium,which was identified as Thelohania solinopsae,Knell,Allen &Hazardby light microscopicanalysis of methanol-fixedand Trichrome-stained smears made from the infected insects. The range of spofe morphotypes,staining patterns, morphology, and measurementswere identical to ?' solenopiie. This is the first evidenceof T, solenopsaein natural infections ofRIFA in Louisiana, The number of RIFA moundsin pastureswith infected ants did not differ from the numberof moundsin pastureswithout infectedants. Only l'6% of moundsin quadrantstreated with methopreneone year before our survey were infected,while the levelof infectionin the otherareas was 15.4'73.3%o' INTRODUCTION Red imported fire ants, Solenopsisinvicta Bwen, cause serious medical and agricultural problems due to their potent sting and large, aggressivepopulations. Damageto pasturesis especiallydifficult to managebecause fire ants are expensiveto control over the large acreageneeded to maintain livestock.When the red imported fire ant (RIFA) establishedin the US, few natural enemieswere brought with them from their native SouthAmerica. The microsporidium Thelohaniasolenopsae I(nell, Allen & Hazard,has been proposedas a potential biocontrol agent for the red imported fire ant. Infection of RIFA queenswlth T. solenopsaecan result in a dramaticdecline in brood productionin both monogyneand polygynecolonies (Williams et al. 1999,Oi and Williams 2002). Briano andWilliams (1997)indicated that f. solenopsaeprogressively destoys the fat body of fire ants,which leadsto shortenedlongevity. The transmissionand life cycle of T. solenopsaeand its relationship to the social structure of ant colonies is not fully understood.Although T. solmopsae is being culhred and introduced into the environmentby the USDA, the organismhas been found outside of releasesites in the southeasternUnited States(Williams et al. 1998). The purposeof this study was to confirm T.solenopsaeinfections in Louisiana, determineprwalence, and describedistribution of infections amongfire ant coloniesin pasturesrelative to previous insecticidetreatments. 27r MATERIALS AND METHODS The study was conductedat the LouisianaState University Agricultural Center Rosepine Researchstation, Rosepine,LA. Five pastures(50A, 5oB, 67,6g'notaturn) and 69), each approximately 2ha and with either bahiagrass (paspalum or bermudagrass(cynodon dactylon), were divided inio four q,radrants. A central circularsample area (36m in diameter)was laid out in eachquadrant, and the antswere collectedfrom this area. A preliminarysurvey was conductedon 3l Jantary 2002, afterwhich multiple sampleswere collected from eachquadrant between 25 Marchand 15 May. A separatesurvey of the perimeterof pasture50A was conductedfrom 5 April to 17April. Samplesof ants were collectedin 20-ml glassvials that were insertedinto each moundwith the mouth of the vial level with the soil surface.To preventthe antsfrom escaping,vials werecoated inside at the openingwith liquid fluoropolymerresin prFE 30 (Teflon, Dupont, Wilmington, DE). After sa4pling, the vials were transportedon ice to BatonRouge, LA , andthen stored at -70"C , In orderto detectmicosporidiosis, 50-100 ants from eachvial werehomogenized in 200u1of steriledistilled water in a 1.5-mlmicrotube with a disposablepestle (Koates Glass9o.,Vineland, NJ). Twenty ul of homogenatewas spreadon a glassslide over 23cm' (areaof a standardcover slip) to obtaina thin transparentlayer without solid particles.Smears were first examinedunder phase contrast optics, air dried,fixed with absolutemethanol for 5 min, and stainedwith modifiedTrichrome stain for 30 min at 37oC(Weber et al. 1992;Didier et al. 1995).Spores were measuredelectronically on methanol-fixedTrichrome-stained smears under 1000X magnificationin bright field with a Nikon EclipseE-600 microscope equipped with epifluorescence,PC anda Meta- View imagingsystem (MetaView 1998).Measurements were comparedto thoseof a known ?. solenopsaeisolate (Sokolova and Fuxa 2001). Occasionally,methanol fixed smearswere stainedalso by Giemsaand CalcofluorWhite M2R (Sigma,St. Louis, MO). on 25 March, the maximum(L) and orthogonal(w) diametersof 20 moundsin pasture50A were measuredand antswere coreJtedfrom eachmound, Mounds areas (A) were calculated as A:til-w/4. Headdiameters of 20 antsfrom eachmound were measured *lt! * Olympus SZXI2 microscope with digital imaging (Media cybemetics 2001). Based upon the ."uru..-.ot., the social structui or tn. *t population was classifiedas monogyne,polygyne, or indeterminate(Greenberg et al. 1985).Between 25 Marchand l5 May thenumber of moundsin p*tu.", soa, soB,67 , 68 and69 wereestimated. In . April 2000 an-d,200r, soyo of the pastureswere treated with broadcast metho-prene(ExtinguishrM professional Fire Ani Bait, wellmark Intl., Bensenville,IL) at 1 kg/ha. The treatrnentswere applied in a manner which resulted in the four quadrants in eachpasture being treatedonce each year, once only in year l, onceonly in year 2, or not h'eatedduring.the 2 years.The quadrantsweri numberedr-4 (e.g., 67.l.was.pasture67 quadrantl), and numbersdid not conespondto prior trearmenq thusblinding the surveypersonnel for treatments. -^- - !L* length and width- comparedby t-test for independentsamples (srATIsrIcA -was for windows 1996). The number of mounds in pastureswith and without infected ants and in quadrantswith and without infected ants was comparedby Tnaire,6 t-test (GraphPad1998). The numberof infectedand noninfectedcolonies (mounds) in pasturestreated with methoprenein 2001vs. pasturesnot treatedin 2001, as well as associationof infectionwith socialstructure, were compared by the Fisher's exacttest (GraphPad 1998). 272 RESULTS The measurements(length and width) of sporesof the previouslydescribed I solenopsaeisolate from Gainsville,FL, andof the microsporidiumfrom antscollected at Rosepine,LA, werecompared (Table l). TABLE.I. sporesMeasurements of z solenopsaec. il andthe Microsporidiumfrom Rosepine(M. R.). Isolate Sporetype Length+SD width + SD N (pm) (pm) Octospores Zs. 3.1+ 0.46 2.1*0.23 7l M.R. 3.1+ 0.29 2.t+0.23 77 T. s. +0.32 Nosema-likespores 4.6 2.36+ 0.21 ll M.R. 4.6r0.28 2.22+0.17 l9 ?. Megaspores s. 6.1f 0.39 3.7+0.25 L6 M.R. 6.2+0.44 3.5+ 0.33 l0 Macrospores(du- T. s. 5.29+ 0.51 2.1+0.25 8 pletsof octospores) M.R. 5.36+ 0.42 2.2*0.21 l4 Sporophorous T. s. 9.16+ 0.39 t4 vesicles M.R. 8.84+ 0.75 l3 The following spore types were found: (i) octosporesgrouped in octets and surroundedby fragile envelopes(sporophorous vesicle, Spv) or unbound,(ii) Nosema- !k9 spores, (iii) megaspores,described by Sokolova and Fuxa (2ooti from a Gainesville,Florida isolateof r.solenopsae,and (iv) "macrospores" dupletsofundivijed o.torpor.r, so called (Knell et al. lg77) which also were enclosedin an Spv envelope or unborurd. .I!" .go*" T. solenopsaevoucher and the Rosepine microsporidiumdid not differ in lengthor width of any of the sporetypes (Tabl; r). The appearanceof fresh sporesunder phase contrast microscope unO ttt" i"tt"- of their stainingwith Trichrome,Giemsa and Calcofluor stains were identical foi ttreRosepine andFlorida isolates. of the 20 moundssampled in pasture50A on 25 March, 7 were positivefor z solenopsae.Three of sevenmounds with indeterminatesocial'structure, two of seven polygyne, andtwo of six monogynecolonies were infected. Moundswitl infectedants sampledon 25 March did not differ in area(n:20, average=ll00cm2+990) or average ant-headwidth (n:20, averagr0.795mm+0.109)from those without infecteoants. Areasof moundswith infectedants were less variable, than those without infectedants (n:20' P=0.02);there were fewcr small(<3l0cm2) and large (>1590cm2) mounds. Ant- head width from mounds without infected ants was [n-arly related io mound area < (R]:0.18' P 0.001),but therewas no rr*il",, rignincantcorrelation for moundswith infectedants. with or without Thelohania,the numberof moundsdid not differ among pasturesnor among quadrantsin infectedpastures (n=g, average=25Omounds/ha + 220). on 31 January 2002, a sampleof ants from one mound in each of the 20 quadrants showedtwo (50A.1 and 68.4)positive for T. solenopsae.Between 25 March and 15 May 2002, multiple sampleswere collected from eachof the quadrants.Ants fiom four of the 20 quadrantswere positive for T. solenopsae,and three of the four positivequadrants had not beentreated with methoprenein i00l (Table2). 273 TABLE 2 Relationof ThelohaniasolenoDsae Infection to MethopreneTreatment Numberof 21s. Methoprene NUmDerot treatment . DOSltlVe mounos samDteo Spring samples Spring 2000 2001 31 Jan Wholearea 22 2 na na 25 Mar 67.2 10 0 no no 67.3 10 0 yes yes 69.4 10 0 no no 69.1 10 0 yes yes 50A,4 10 1 yes yes 504.1 10 6 no no 5 Apr 50A.3 10 0 no yes 50A.2 10 6 yes no 508.4 10 0 no no 68.2 10 0 yes no 68.1 10 0 no no 68.3 3 0 no yes 68.4 10 0 yes yes 15May 508.1 5 0 yes yes 508.2 3 0 no yes 50B.3 5 0 yes no 67.1,67.4 6 3 no no 69.2 3 0 no yes 69.3 3 0 yes no Ants from 12 of 78 mounds(15.4%) sampled in quadrantsthat werenot treatedin 2001were positive for T. solenopsae,whileants from only oneof 64 mounds(1.6%) in quadrantsthat were treatedin 2001 were positive for T. solenopsce.Thus, methoprene- treatedmounds had a lower prevalenceof infection(P = 0'001, df =1, X2 = 8.1).An untreated,triangular corner area adjacent to pasture50A andalongside a roadcontained infectedcolonies (ll of 15 or73.3Yo),butnoinfected ants were foundin 13 mounds sampledin an untreatedarea across the road from pasture50A' DISCUSSION The rangeof sporemorphotypes, staining patterns, morphology, width and length' as well as hoit speciesand tissuelocalization, were identicalbetween T. solenopsae and the newly dlscoveredmicrosporidium at Rosepine,sfrongly indicatingthat the microsporidium found in ants collected at Rosepine is T. solenopsae.Although L solenopsaeis widespread in Florida and Texas (Williams et al. 1998)' our results representthe first detectionofnatural infectionsof S. invictaby T. solenopsaein the stateoflouisiana, T. solenopsaeis virtually absentliom eastemLouisiana except at sites where it has been introducedexperimentally (Sokolova and Fuxa, unpublished data). Infection mtes of colonies in Louisiana were similar to those in Argentina, where prevalenceaveraged 8o/o and was as high as 80% (Briano et al' 1995a). Similariy,T. solenopsaeprevalence rates were 37o/o in Mississippi,73% in Florida,and prevalenceand 7l%oin texas, lWiitlami et al. 1998).Determination of this baseline distributionis'nicessary before releasing this microsporidiumfor biologicalcontrol in westernLouisiana. Wiliiams et al. (1998)suggested that T.solenopsae infections would 274 be found only in polygynouscolonies, but our findingssupport reports ofmonogynous colonieswith infectedants (Briano et al. 1995b). Sincethe prevalenceof T. solenopsaeinfections ofants in the samplequadrants was not determinedbefore the methoprenetreatments were applied,we cannotassess the treatnent effect on the prevalenceofinfection in the different quadrants.However, our observationthat the infection rate was 15.4o/ofor colonies in areasnot treatedthe year before and 1.6% for coloniesin areastreated with methoprenethe year beforemay indicate faster elimination of the infected moundsdue to treatments.This phenomenon deservesfurther investigation. Z solenopsaecan weakericolonies in conjunctionwith insecticidetreatment (Oi and Williams 2002). Regardingthe distribution of 11 solenopsae,it was interestingto frnd 73Yoinfection for samplescollected around the previously untreatedperimeter areasof pasture50A, while none of the samples collectedacross the road were infected. It is possiblethat a well-traveled,5-m-broad roadmight reducethe overlapofforaging individualsfrom adjacentcolonies. ACKNOWLEDGMENT We thank L. Bui, K. Heyer, A. MacKay, A. Richter and M. Seymore.The researchwas supportedby a special researchgrant from the LouisianaBoard of Regentsand by USDA RegionalIPM grant#99-34103-8067. This paperwas approved for publicationby the Director of the LouisianaAgricultural ExperimentStation as manuscriptno. 3485. LITERATURECITED Briano, J. A., D. P. Jouvanaz,H. Cordo, R. Patterson.1995a, protozoan and fungal diseasesin solenopsis richteri and s. quinquectupis(Hymenoptera: Formicidae) in BuenosAires Province,Argentina, Florida Entomol. 78: 531-537. Briano,J. A., R. Patterson,and H. cordo. 1995b.Relationship between colony size of solenopsis richteri (Hymenoptera:Formicidae) and infection with Thelohania solenopsae(Microsporidia: Thelohaniidae)in Argentina, J. Econ. Entomol. 88:1233-1237. Briano, J. F., and D. F. williams. 1997.Effect of the microsporidiumTherohania solenopsae(Microsporida: Thelohaniidae) on the longevity and survival of Solenopsis richteri (Hymenoptera: Formicidae) in the laboratory. Florida Entomol.80:366-376. Didier, E. S.,J. M. Orenstein,A. A. Aldras,D. Bertucci,L. B. Rogers,and F.A Janney. 1995.Comparison of threestaining methods for detectingmicrosporidia in fluids. J.Clin. Microbiol.33: 3138-3145. GraphPad.1998. Instat, version3.00 for windows 95. GraphpadSystems, San Diego cA. Greenberg,L., D. J. c. Fletcher,and s. B. vinson. 1985.Differences in workersize and mound distribution in monogynousand polygynouscolonies of the fire ant SolenopsisinvietaBwe* J. KansasEntomol. Soc. 58: 9-18. Knell, J. D., G. E. Allen, and E. l.Hazard. 1977.Light and elechonmicroscope study of Thelohaniasolenopsae n. sp.(Microsporida: Protozoa) in the red importedfire ant,Solenopsis invicta. J. Invertebr.Pathol. 29: 192--200. Media cybernetics.2001. Image-Pro Plus version 4.5.0.19.Media cybernetics,silver SpringMD. 275 MetaView. 1998. Meta Imaging Series4.5, Universal Imaging Corporation,West ChesterPA. Oi, D.H. and D. F. Williams. 2002. lmpact of Thelohaniasolenopsae (Microspridia: Thelohaniidae)on polygyne colonies of red imported fire ants (Hymenoptera: Formicidae).J. Econ.Entomol. 95: 558-62. Sokolova,Y. Y. and J. R. Fuxa. 2001. Developmentof Thelohaniasolenopsae inred imported fire ants Solenopsis invicta from polygynous colonies results in formationof threespore types. J. EukaryoticMicrobiol.,48 (Suppl.):85S. STATISTICAfor Windows1996, release 5.1, StatSoft inc. 1994-1996.V 5. Weber,R., R. T, Bryan, R. L. Owen, C. M. Wilcox, L. Gorelkin and G. Visvesvara, 1992.Improved lighfmicroscopic detection of Microsporidaspores in stool and duodenalaspirates. New EnglandJ. Medicine4: 16l-166. Williams, D. F., G. J. Knue, and J. J. Becnel. 1998.Discovery of Thelohania solenopsaefrom the red importedfire ant,Solenopsis invicta, in the UnitedStates. J. Invertebr.Pathol. 7l :175-176. Williams, D. F., D. H. Oi, and G. J. Knue.l 999. Infection of red importedfire ant (Hymenoptera:Formicidae) colonies with the entomopathogenThelohania solenopsae(Microsporidia: Thelohaniidae). J. Econ.Entomol. 92: 830-836. 276 vol.29 NO.4 SOUTIMESTERN ENTOMOLOGIST DEC.2004 DEVELOPMENTOF A SEQUENTIALSAMPLING PLAN FOR CABBAGE LOOPER, TNCHOPLUSIA ffI(Hiibner) ECGS IN COMMERCIAL CHILI PEPPERS Mark A. Muegger ABSTRACT A field study was conductedto develop a sequentialsampling plan for estimating cabbagelooper egg (cLE) densityin commerciallyproduced chili peppers.Results from this study indicatedthat sampleunit size did not influencespatial pattems exhibited by cabbagelooper (cL) oviposition;however, Taylor's power law model fit the l- and 5- plant sampleunit size databetter than the 2-,3-, and l0-plant sampleunit size data. All plant samplesizes were usedto developGreen's fixed precisionsequential sample plan. Cost reliability data indicatedthat the 5-plant sampleunit size was the most efficient sampling method. simulation of the sampleplans using the RSW program of Naranjo and Hutchision (1997) indicatedthat all models fit the sample data well, but acfual precisionlevels were lower than the desiredprecision level for high cabbagelooper egg densities. The 5-plant sampleplan was optimized using the RSVp prograrnto reflect thi desiredprecision level, optimization resultedin a >20%oreduction in samplingeffort relativeto thenon-optimized sample plan. INTRODUCTION . Numerousinsect pestsoccurring in the United Statescan causesignificant yield loss in chili peppers. Those of primary concern include com earworm-,Helicoverpa zea @o{die), beet armyworm, Spodoptera exigua (Hibner), and the pepper weevil, Anthonornuseugenii cano. other occasionalpests may also cause ecbnbmic loss in peppers. one of these,the cabbagelooper (cL), Trichoplusiani (Hiibner), causes extensivedamage to manyvegetable crops (Soo Hoo et al. 19g4). The larval stageof this lepidopterousinsect pest causeseconomic damage by feeding on the plani foliage. Extensive larva feeding initially causes a shot-hole appearanie that may eventuaily proceedto partialor completedefoliation ofthe plant. Methods for efficiently and reliably monitoring pest populationsare central to integratedpest management(IPM) theory. Cunently, several methods exist for sample plan development.These methods result in eitherfixed samplesize or sequentialsamile plans'The numberof samplesrequired to estimatepest population densitid usingGreen's (1970) or Kuno's (1969) fixed precisionsequential sampling plans varies ivittr pest fgnsity, thus reducingrequired sample numbers when pest population densities are either high or low. Fixed samplesize plans,on the other hand, require a fixed numberof samples to estimate pest density regardlessof pest density and are generally not as efficient as sequentialsampling plans when makirg pest managementdecisioni (Nyrop andBinnsl99l). consequently,sequential sampllng plans have seenextensiveusein pestmanagement (Fowler and Lynch l987a,b). rTexas CooperativeExtension, Fort Stockton,TX 79735 277 SeveralMonte Carlo approachesfor validatingsequential sampling plans have been developed(Naranjo and Hutchison1997, Trumble 1989,Nyrop and Binns 1991,Binns 1994). As Naranjoand Hutchison(1997) point out, thesevalidation methods are limited by drawingobservations from theoreticaldistributions or empiricalmodels and may not reflect actualfield collecteddata. Their validationprogram (resampling validation for samplingplans) usesreal field data to validate samplingplans and thus obviatesthe intrinsicproblems associated with validationmethods based on theoreticaldistributions or empirical models. The purposeof this study was to developa reliable and efficient sequentialsampling plan for cabbagelooper eggs (CLE) in commerciallygrown chili peppers. MATERIALS AND METHODS A total of eight commercialpepper fields locatednear the fuo Granderiver in El Paso County,TX, weresampled for eggsof variouslepidopterous pests in 1999. Fieldsranged in size from approximately l0 to 40 acres. Fields were visually stratified into four quadratesso that samplesrepresented all portionsofthe fields. Randomlyselected fields weresampled twice weeklyfor I I consecutiveweeks. Sampleswere obtained by walking in a quadratefor l5s and selectingthe nearestplant as the startingpoint for the sampling bout. Each samplingbout consistedof l0 consecutiveplants in a row and was timed using a stopwatch. Samplingconsisted of whole plant visual inspectionof eachplant. Five samplingbouts were taken in this mannerfrom eachof four quadratesin the field for a total of 20 samplingbouts per field. Collecteddata were divided into two data sets. One data set was usedto developthe sampleplan, while the other data set was usedto validate and optimize the sampleplan. Collecteddata for both data sets were later partitionedinto 1,2,3, 5, and 10 plantsper sampleunit for determinationof optimum sampleunit size. Spatialstatistics for eachsample unit sizewere determinedfrom Taylor's PowerLaw by regressingthe natural log of the varianceagainst the natural log of the mean' The slope(p) of the regressionline is an indicatorof aggregationand the intercept(ct) is a samplingfactor (Taylor 196l). To evaluatethe fit of Taylor's aggregationmodel to the field data; expectedand observedvariances were computedfor each sampleunit size standardizedto common mean CLE densities and regressionanalysis performed. Analysisof variancewas performed on estimatedvariances obtained from Taylor'spower law for eachsample unit sizestandardized to commonmean CLE densitiesto determineif the aggregationcharacteristics cr, p, and cost reliability significantly differed among samplesizes. Meanseparation was performed using Fisher's protected LSD. Green's(1970) fixed precisionsequential sampling plan was developedfor the 1-, 2-, 3-, 5-, and l0-plant samplesizes. The samplingstop line was determinedfollowing the equation: * LNT)= [LN(8 /d )(B -2)] + t(B - I /B -2) LN(n)J where 4:tbe cumulative number of individuals, n=the total number of samples,D=the fixed precisionlevel (SE/meanito.osn:|.96), and g and p me the reglessionparameters of Taylor'sPower Law. Samplingplans were evaluatedand optimizedusing a resamplingapproach program (resamplingvalidation for samplingplans) (Naranjo and Hutchison1997). Data setsfor sampleunit analysiswere taken from one randomlyselected field from eight fields per week, Randomlyselected fields were sampledover 12 consecutiveweeks (one sample containedno data and was not included)to give a total of ll data setsfor eachof the sampleunit sizes. The resamplingprogram randomly selectsvalues from a specifieddata 278 set until the mean density is estimatedat the specified precision level and minimum sample number. This procedure was repeated500 times for each data set and plant sample unit size. Data generatedfrom the resampling program included actual and observedmean densities, minimum and maximum samplenumbers needed to make a pest managementdecision, mean precisionlevels, and minimum and maximum precision levels. Economicthresholds are currently not availablefor CLE in peppers,thus comparisons of samplingplans were madeusing an arbinarily selectedCLE meandensity. Sampling plans were optimized by varying the precision level and minimum sample numbers neededto estimatemean egg densities. Relativeefficiencies of samplingplans were determined by usingcollected sample and walking time data for each of the sample unit sizes evaluatedapplied to Wilson et al. (1982)cost reliability equation.Cost reliabilities were determined by: Ct/Cz=n (st t + wt)/n2(st2 + wt) whereCrthe total cost(in time) to estimatea specifieddensity and precision level for the sampleplan, n,:the number of samplesrequired, s/Ftime required to take a sample,and wtptime required to walk to the next samplearea. Values less than one indicate that the numeratorsampling plan is more efficientwhile valuesgreater than one indicatethat the denominalorsampling plan is more efficient. RESULTSAND DISCUSSION Coefficientsofdetermination for eachofthe plant samplesizes indicate that a shong mear/variancerelationship exists regardlessof plant sample size; however, that relationshipappea$ to be strongestfor the one-plantsample size (Table l). Theseresults indicatethat the oneplant samplesize should be a betterpredictor of meanCLE densities relativeto the otherplant samplesizes. TABLE l. AggregationCoefficients and Associated Statistics for CabbageLooper Eggs at EachPlant Sample Size. Plants/SampleUnit I 0.808 0.937 0.78 0.0001 2 0.819 0.955 0.69 0.0004 5 0.767 0.937 0.67 0,0006 { 0.785 1.013 0.71 0.0003 l0 0.842 0.989 0 0.0023 Based on aggregationcoefficients from Taylor's power law, estimatedvariance to meanrelationships were sigrificantly different amongthe plant samplesizes (Table 2). These results could indicate that spatial pattems of CLE oviposition were significantly influencedby the plant samplesizes used in this sfudy. However, the sampledata in this studywere partitioned to obtainthe differentplant,sample sizes and thus did not represent independentdata sets. wilson (1994)points out that when sampledata arepooled from consecutivesamples to obtain different sample uriit sizes, Taylor's coefficients would "biologically inqreaseor decreasedepending on the relevantsample unit." Taylor's coefficientsfor the different sampleunit sizesin this study did not appearto follow a "biologically trend that would provide any information regarding the relevant sample 279 unif' for CLE (Table l). Interestingly,however, all but the l0-plant samplesize resulted in Taylor's coefficientsthat gave estimatedvariance to mean relationshipsthat were significantlyless than onefor all plant samplesizes except the l0 plant samplesize (Table 2). This resultsuggests that the spatialpatterns exhibited by CLE on the l-, 2-, 3-, and5- plant samplesizes were more uniformly distributedwhile CLE on the l0-plant sample size was disfributedamong the l0-plants at random. Once again,however, this result couldbe an artifactofdependent data sets, sampler effrciency, random sample variability, and/orother factors (Wilson 1994). TABLE 2.Mean Variance/MeanRelationships of CabbageLooper Eggs for EachPlant SampleSize in CommercialChili Fields. Plantsample size Variancet /mean ratioo I 0.83294b 2 0.83739b 3 0.79102c ) 0.78055c l0 l.0l79la Yariance estimatedfrom Taylor's coefficientsacross standardized mean cabbage looperegg densities.bMeans followed by the sameletter are not sigaificantlydifferent (Fisher'sprotected LSD; P:0.05). To determinethe meancost reliability of eachof the plant samplesizes, Green's fixed precisionsequential sampling plan was developedfor the l',2',3-,5-, and l0-plant samplesizes across mean CLE densitiesranging from 0.1 to 2.0. Mean cost reliability sigrrificantly differed amongplant samplesizes (Table 3). The l0-plant samplesize was significantlyless cost efficientthan all other samplesizes, while the 3-plantsample size wasthe most cost efficient. TABLE 3. CostReliability of the 2-, 3-, 5-, and l0-Plant SampleSize Plans Relative to the one-PlantSample Size Plan Across a Rangeof cabbageLooper Egg Densities. Plantsample size 0 CostReliabilitf I 1.00b 2 0.93b J 0.85c 5 0.95b l0 l.l8a atues lt indicate more cost reliable; values>l indicate cost reliable. Cost reliability determinedfrom mean cabbagelooper egg densitiesrang]nq from 0'1-2.0. Meansf:ollowed by the sameletter are not sigrrificantlydifferent (Fisher'sprotected LSD;P:0.05). Validation of the samplingplans illustrate the stochasticnature of Green'sfixed precisionsequential sampling plan (Table4). The stochasticnature of thesesample plans iu* b"ro pieviously documented and was expected (Naranjo and Hutchison 1996, Hutchisonl9g+, ttutcUisonet al 1988). Reasonsfor variability of sequentialsampling plansare given by Hutchison(1994) and Nyrop andBinns (1991) and need not be covered Lere. ThJmost important purposeof sampleplan validation is to determinehow well the samplingplans periorm and to optimizethe sampleplan if necessary.The sampleplans 280 developedin this study performed well with actual precision levels varying little relative to the desiredprecision level (actualprecision levels for all sampleunit sizesample plans precisionlevels tendedto be slightly lower for the low cLE populationdensities and rangedfrom 0.168to 0.178;desired precision was set at0.179).Actual averageprecision precision levels tended to be slightly lower for the low CLE population densities and levelsin generalwere slightly betterthat the desiredprecision level. Interestingly,actual precisionlevels tendedto be slightly lower for the low CLE populationdensities and higherfor thehigh CLE populationdensities. One explanation for theseresults, at leastin part, could be causedby the minimum samplesize restriction placed on the larger plant samplesize sampleplans. At high populationdensities the minimum samplenumber would havebeen lower thanthe selectedminimum sample number for the 5- and l0-plant sarnplesize sampleplans. Thus, more sampleswere taken than neededto estimatethe high population densities.This explanationdoes not, however, explain why actual precision levels tendedto be lower at the high populationdensities for the smallei sample unit samplesize sample plans. TABLE 4. Resultsof RSVP Simulationof the 3-Plantsample plan for Estimatingper Plant Cabbagelooper Egg Density before Optimization @=0.179 and Mimimum SampleSize5). Observe Est 0 Max Min d 0 CLE 0 CLE Precision Precision Precision 0 Max Min Density Density Level Level Level Samp# Samp# Samp# 0.1 0.13 0.34 0.35 0.32 75 184 23 0.t2 0.l5 0.38 0.50 0.32 65 160 13 0.24 0.27 0.33 0.35 0.19 29 689 0.39 0.43 0.31 0.44 0 l8 477 0.66 0.75 0.35 0.53 0.11 11 2s5 0.79 0.86 0.29 0.48 0 9 195 1.64 1.68 0.26 0.58 0 5 95 r.82 t.92 0.44 r.00 0 6 135 2.63 2.67 0.22 0.46 0 5 65 2.82 2.87 0.17 0.41 0 ) 55 comparison . of sample.plans, as previouslystated indicatedthat the 3-plant sample size sampleplan gavesignificantly better cost reliability relativeto all othersample sizes. Thus the 31lant sampleplan was chosento validateand optimize. Becausethis sample plal yas being developef fo1 an integratedpest managementscouting program, optimization was conducted.todecrease precision at the higtierpopulation densities that would be important for making a pest managementdecisioi. Estimating low population densities may be usefulfor researchpurposes; thus, the non optimizedsample plan could be usedfor researchpurposes. optimization of the 3-plant sample plan was conductedby running the RSVp simulation p.logram using higfue1desired precision levels and lower miniirum sample numbers until actualachieved precision was nearest the originaldesired precision level of a=0.179. A final precisionlevel of d=0.20and a minimim samplesLe oi threewere chosen. Results of the optimization are presentedin Table 5. The actual average precision level for cLE densitiesgreater than 0.15 per plant afteroptimization was 0.171. when Green'sfixed precisionsequential sample plan for cLE was optimized,the required samplesize was reduced,relative to the non-optimizedsample pian, by ioyo o, ^or" (Table6). 281 TABLE 5. Resultsof RSVP Simulationof the 3-PlantSample Plan for Estimatingper Plant CabbageLooper Egg Densityafter Optimization(D=0.2 and Mimimum Sample Size=3). Observ--::' Est 0 Max Min ::: 0 cLE Precision Precision Precision 0 Max Min ^tt: Densitv Level Level Level Samp# Samp# Samp# uenslty - o.l 0.i3 0.39 o.4o 0.31 s7 166 13 0.r2 0.15 0.43 0.57 0.34 50 133 6 0.24 0.28 0.36 0.39 0 25 61 5 0.39 0.44 0.34 0.48 015424 0.66 0.77 0.40 0.68 09243 0.79 0.87 0.34 0.66 07163 1.64 1.75 0.31 0.81 047 1 1.82 2.12 0.51 1.00 0 4 ll 3 2.63 2.62 0.27 0.72 0353 2.82 2.84 0.20 0.63 0343 TABLE 6. Green'sFixed PrecisionSequential Sampling Plan for CabbageLooper Eggs before and after optimization using the Resamplingvalidation for sampling PlansProgram. BeforeOptimization (D=0. 179) Aft er Optimization(D=0.20) Cumulative Sample Cumulative Sample 0 CLE/plt CLE Number 0 CLE/plt CLE Number ')\ 0.18 45 0.1 l9 40 0.2r 25 40 0.18 t9 35 0.23 25 35 0.2r l9 JU 0.27 24 30 0.25 l9 25 0.32 24 25 0.31 l9 20 0.40 24 20 0.32 l8 l9 0.42 24 19 0.34 l8 l8 0.44 24 l8 0.36 l8 t7 0.46 24 t7 0.38 l8 l6 0.49 23 l6 0.40 l8 l5 0.52 23 t5 0.43 l8 l4 0.55 23 t4 0.46 l8 13 0.59 23 l3 0.50 18 12 0.64 23 l2 0.54 18 ll 0.70 23 ll 0.59 18 10 0.76 23 10 0.65 l8 9 0.84 23 9 0.73 18 8 0.94 23 8 0.83 l7 7 1.06 22 0.96 l7 6 1.23 22 6 l.l4 t7 ) 1.46 22 5 1.40 l7 4 1.80 22 4 1.84 t7 3 2.36 2l 282 The resultsfrom this studyprovided a reliableand effrcientsequential sampling plan suitablefor use in an IPM progrzlm. Sequentialsampling plans, however,are rarely followed by scouts,consultants, and producersbecause of the addedeffort involved in conductingsequential sampling in the field. Choosingthe lowestmean density that would be importantfor the pest speciesin questionand conductingall samplingat the required samplenumber for that density(fixed sampleplan) could alleviatethis problem. This samplingmethodology may solvethis problembut would substantiallyincrease sampling effort when pest population densitiesare either high or low. Nevertheless,use of samplingplans to estimatearthropod pest densitieswould result in more precisetiming of pesticide applications. Further, use of sampling programs would result in more environmentallysound and cost effective productionpractices. LITERATURECITED Binns, M.R. 1994. SequentialSampling for ClassifyingPest Status.pp 137-174.In L.P.Pedigoand G.D. Buntin [eds.].Handbook of samplingmethods for arthropodsin agriculture.CRC, Boca Raton, FL. Fowler, G. W. and A. M. Lynch. 1987a.Bibliography of sequentialsampling plans in insect pest managementbased on Wald's sequentialprobability ration test. Great LakesEntomol. 20: 165-171. Fowler,G. W. andA. M. Lynch. 1987b.Sampling plans in insectpest management based on Wald'ssequential probability ratio test. Environ.Entomol. 16:345-354. Green,R. H. 1970.On fixed precisionlevel sequentialsampling. Res.Popul. Ecol. 12: 249-251. Hutchison,W.D. 1994.Sequential Sampling to DeterminePopulation Density, pp 207- 243. In L.P. Pedigo and G.D. Buntin [eds.]. Handbookof samplingmethods for arthropodsin agriculture.CRC, Boca Raton,FL. Hutchison,W.D., D.B. Hogg, M. A. Poswal,R. C. Berberet,and G. W. Cuperus.1988. Implications of the stochasticnature of Kuno's and Green's fixed-precisionstop lines:Samplingplans for the pea aphid (Homoptera:Aphididae) in alfalfa as an example.J.Econ. Entomol. 8l: 749-758. Kuno, E. 1969.A new methodof sequentialsampling to obtainpopulation estimates with a fixedlevel ofprecision. Res. Popul. Ecol. l1: 127-136. Naranjo,S.E. and W.D. Hutchison. 1997.Validation of arthropodsampling plans using a resamplingapproach: software and analysis. Amer. Entomol. 43;l pp48-52. Nyrop, J. P., and M. Binns. 1991.Quantitative Methods for Designingand Analyzing SamplingPrograms for use in PestManagement, pp, 67-132. .In Hanson,A.A., and D.Pimentel[eds.], CRC Handbookof PestManagement in Agriculture. CRC, Boca Raton,Fla. Soo Hoo, C.R., D.L. Coudriet, and P.V. Vail. 1984 Trichoplusianl (Lepidoptera: Noctuidae)larval developmenton wild and cultivatedplants. Environ. Entomol. 13: 843-846. Taylor,L. R. 1961.Aggregation, variance, and the mean.Nat. 189:732-735. Trumblg J.T., Brewer,M.J., Dhelton,A.M., andNyrop, J.P. 1989.Transportability of fixed precisionlevel samplingplans. Res. Popul. Ecol. 3l:325 Wilson,L.T.1994. EstimatingAbundance, Impact, and Interactions Among Arthropods in Cotton Agroecosystsems,pp 475-514. In L.P. Pedigo and G.D. Buntin [eds.]. Handbookof samplingmethods for arthropodsin agriculture.CRC, Boca Raton,FL. )9,? vol.29 NO.4 SOUTHWESTERNENTOMOLOGIST DEC.2004 PEST MANAGEMENTPRACTICES IN STOREDPEA}IUTS IN TI{E SOUT}I\ilESTERN TINITED STATES DudleyT. Smith Departmentof Soil andCrop Sciences TexasA&M University CollegeStation, T exas7 7 843 -247 4 [email protected] ABSTRACT Surveys were conducted in 1998 urd 2O03 to assesspest problems, management practices,and pesticideuse in the storageofthe southwestempeanut crop, which accountsfor 26%oof total U.S. production. Shellersand warehouse operators employed sweral integrated pest management(IPM) methods for insect pests in stored peanuts. IPM strategiesof prevention, avoidance,monitoring, and suppressionincluded severalphysical and chemical tactics. All firms followed written Good ManagementPractices and site cleanup procedures prior to storage. Prophylacticgeneral surface and crack and creviceinsecticides were applied by 83%oof the firms. Scoutingwas widely practiced,withT6Yo of the programsusing grain probepit fall traps. Extensivemonitoring and scouting reduced the useofautomatic scheduling of insecticidetreatments. All firms appliedinsecticides in the springas seasonaltemp€f,atures increasedand insectsbecame more prwalent in the remainingunshelled commodity. Changes in insecticideuse were notedbetween 1998 and 2003 in pre-storage,top dress,head space, and fumigation treatments. In the southwesternU.S., 18% of the total in-shell peanutcrop was treated in storage. Vertebratepests included birds and rodentsbut did not pose significant problemsfor controlin peanutwarehouses. INTRODUCTION Assessmentsof pestmanagement practices and pesticide use in agriculturalcommodities havefocused primarily on crop protection in the field and not the subsequentstored products. Howwer, losses due to pests in stored crops frequently equal or exceed those in field production. Peanuts(Arachis L.) are grown on more than 160,000ha in Texas, OHahom4 and New Mexico in the southwesternU. S., which represents26% of the U.S. production. In contrastto other peanutproduction areasin the U. S., this region producesall four market types of peanut(runner, Spanish"Virginia, and Valencia). Runnertlpes are high yielding , accountfor 64Yoofthe productionin the southwesternstates, and are predominately usedfor peanutbutter. Spanishpeanutsmakeup22%o ofthe production,poss€s a reddishskiq andare used in peanutbutter andconfectionary products, such as salted nuts. Virginia varieties, with theirlarge nuts, make up 8% of theproduction and are commonly consumed as roasted in- shell"ball parld'peanuts,due to the largekernels. SeveralValencia peanut varieties, grown in New Mexico, makeup 6% ofthe southwesterncrop andhave three or morekernels per shelland are boiled or roastedand sold as an in-shell product, Peanutsin the southwesternU. S. are grown under irrigation in a region where low rainfall and low relative humidity prevail. The semi-arid conditions of this region have reducedthe dependenceon pesticide use in field production(Smith et al. 1998). 285 "farmer After harvest, stockpeanuts", peanuts that havenot beenshelled, crushed, or cleaned,are dried in-shelland storedin warehousesfrom oneto ten monthsuntil shellingis completed.During this storageperiod, several insect pests, birds, and rodents pose a threatto in-shellfarmer stock peanuts.Insect pests can changemore rapidlyand imposefar greater quality lossesthan anyother type of pest(IIagstrum andFlinn 1995). After shellhg kernelsare typically placedin refrigeratedstorage until shipmentto a manufacturerfor further processing into numerousfood products. Peanutbutter is the primary product, wherequality is of utmost concern. Numerousstored product insect p€sts oommonly infest farmer stock peanuts. Redlinger and Davis (1982) describednumerous post-harvest pests in peanuts,with key pestsbeing Indianmeal moth [Plodia interpunctella (IIUbner)], red flour beetle fTribolium castaneum Slerbst)1,grainbeetles(Oryzaephilas spp.),lesser grunborerlRlryzoperthadominica(F.)1, and almondmoth [Cadra cautella (Walker)]. Thesepests tend to existas a guild ofinsectsthat feed on damagedor looseshelled kernels. Economic losses occur from bothphysical destrustion and contaminationfrom frassand castings. Insectpopulations in storedproducts tend to be more prevalentin areaswith higherambient temperatures and humidity but lossescan be suppressed by using forced ambientair to reducetemperatures (Noyes et d. 1995). Integratedpest management(IPM) in stored food products dependsheavily on sanitation,population monitoring, and chemicalcontrol Slagstrum and Flinn 1995). Severalnon-chemical control methodshave been explored to reducelosses to insects.Sanitation is an importantstrategy. Cleaningoffarmer stockpeanuts at harvestto removeloose shelled kernels, foreign material, andother feeding sources may reduce insect damage during storage (Arthur 1989). Historically,insecticides, insectgrowthregulators(IGR), desiccants, andfumigantshave beenused to reduceinsect damage. More recently,biological control agents have been partially effectiveagainst some pests. For example,diatomaceous earth (silicon dioxide as an inert dust) hasbeen evaluated in laboratory-scaletrials (Arthur andBrown 1994). Finely ground diatoms causedeath by dessicationand abrasionof larya. Indianmealmoth and almond moth have exhibitedhighly variableresp onsesto Bacillus thuringiensis@t) (Arthur andBrown 1994and Kinsingeret al. 1980). Trichogramma(7. pretiosumNley) has shown potential as a biocontrol agentinstoredcrops@rower1983).RedlingerandDavis(1982)summarizedtheuseofnatural parasites,predators, and pathogensin post-harvestpeanuts but noted that commercial augmentationand introductions ofviruses, bacteria, protozoans, and other natural enemies had notbeen adopted. Surveillance sampling methods included visual inspections in storagefacilities and the use of grain probe pitfall traps and pheromones. Insecticideshave been shown to effectivelycontrol insects in storedpeanuts (Arthur 1989). Previously,pre-plant fungicide seed treatments and IPM practicesin thefield production of peanutsin the southwesternU.S. were assessed(Smith et al. 1998, Smith et al. 2000). Surveys have been reported on insectsin stored grain (Corral et al. 1992) but except for a generalizedsuney (Anon. 2001), little has been publishedrecently on post harvestpest managementin peanuts.For a more adequateassessment of all pestmanagement practices and pesticideuse in peanuts,a formal nssessmentofpost harvesttactics was needed. The purposeofthis study was to assessinsect and vertebrate pest problems in post- harveststorage and to describepest control practices employed in the peanutindustry in the southwesternUnited States. APPROACHA}.ID METHODS A surveywas initiated in 1998and repeatedin 2003 with six peanutshelling firms who collectivelypurchased, stored, and processed 98olo ofthe total peanutcrop in the southwestem region,that includesTexas, Oklahoma, and New Mexico. Four ofthe six firms operatedin two or more statesand three of the fifms handledall four markettypes. All participatingfirms had 286 points,driers' well-establishedcorporate procedures for the operationoftheir numerousbuying in the region' ti"*g" "iitr, *A processingfacilities, which wire tocatedthroughout 3 5 counties n"gi"""l -i f""i facility managerswere exp€ctedto follow theseprocedures. A surveyinstrument was=prepared and pre-testedwith shellerrepresentatives to assure aspectsof that proper questionsanA terminotoiy would bi usedin obtaininginformation on all Shellers insect and vertebratepest managemintstrategies and tactics. The SouthwestPeanut participate Associationassisted in identii'in! ageneralmanager or centraloperations managerto 1998' in the suney. Theserepresentatl'es utere interviewed by phoneor in person-inFebruary' assess iil;;.!,;". repeatedfive yearslater, in April, 2003, to confirm earlierfindings and Food a"V t"oAn""tions of practicet C,r" to ohangesin pesticideavailable or impac{sof the partioipated in the 1998 and 2003 e,iJitv ptot"r,ion Act (FePA). All firms wi[ingly loth ti*ry*. Three of the respondentsparticipated in both sumeysand, due to retirements,thfee new iepresentativesparticipated in the 2003 sumey but were familiar with the earlier pest management- procedures in their company. The suweyincluded questions on managers'ranking ofinsect pests,pre'storage clean up and sanitatiotrmeasures,methods for removingpeanut debris and harboragefrom storage f""ititi"r (air, sweeping,hosing, and other methods),internal inspections and supervisionprior tostorage,in-storagemonitoringactivities(whenconducted, how, who conducted,trappingand surveillirce methods,and thresholds or basisfor action), control measures(chemical and non- chemicalmethods, and chemicalselection factors), and questionsabout avianand mammalian pestsin storagefaiilities. Definitive responseson objectivequestions were numerically recorded andnotes were recordedon open-endedand subjective questions. One condition ofthe shellers' participationwas that resultswould be aggregatedto protectproprietary business information andpractices. RESULTSAND DISCUSSION Insect Pests. All warehouseoperators demonstrated a working knowledgeof insect pests,could articulatepest management practices and chemical approaches for control, andwere generallyaware ofthe costsand alternatives for control. Whenasked to commenton the relative i-mportanceofinsect pests,all managersmentioned Indianmeal moth asthe mostserious pest and 6?% mentionedthe red flour beetle as another key pest at all locations in the southwest. 'Seetles in general"were mentionedby all participants,without regardto anyparticular species. One operator,particularly skilled in pest identification,mentioned the sawtoothedgrain beetle [Oryzaephilussurinanensis (L.)]as an occasionalpest. Therewas no mentionofthe merchant grain beAle lOryzaephilusmercator (Fauvel)] or almondmoth. Mention of thesepests was ii.ilar to those reported earlier by Redlhger and Davis (1982). The imported fire ant (Solmopsis irwictoBurur) was mentionedas a nominalproblem around the exterior of some storagefacilities and cockro aches(Blatella germanica andPeripalneta americanaLinnaeus) wereoccasional pests at one site. Responsesto specificand open-ended questions on pestcontrol practices followed well- recognizedIPM strategies.ThePAMS (Prevention,Avoidance, Monitoring, and Suppression) modil dweloped by Coble(1998) was employedto summarizesurvey responses in a systematic manner. Major strategicsand tactics are summarizedin Table l. Prevention. Oncepeanuts are dried and placed in storage,physical accessibility to insects is restrictedand control options become more limited. All managersrelied heavily on pro-active sanitationmethods before the commoditywas placedin storageto minimizeinsect infestationslater. HACCP (tlazard Analysisof Critical Control Points) proSrams,which are commonthroughout the food processingindustry, were beingfollowed by five of the six firms. Shellersreported that HACCP plans had been developedand implementedwith cotnmon agre€ment with buyers and food manufacturers. These preventive action plans included 287 measuresto avoid problemsof insect contaminationand quality assurancemeasures 'rsers. for end- Warehousemanagers reported that buyerrepresentatives visited various peanut storage sitesat leastannually to rwiew andaudit HACCP complianceas part ofthe food manufacturerls food safetyprogram with U.S. Food and Drug Administrationind USDA standardssince the post-harvestcommodity must be handledin compliancewith food-qualitystandards. TABLE I . SummaryofPAlvfS @revention,Avoidance, Monitoring, andSuppression) Practices in the Storageof Farmer-StockPeanuts in Texas,oklahoma, and New Meiico in 199g. Strategy/tactic Firms usinglractice ("/") Prwention Haveand use a NACCP pro-activesanitation plan 83 Use peanutindustry GMP plansand practices 100 Avoidance- prior to storage Siteclean up 100 Mechanical- brush,sweep, air 100 Water/washdown 33 Managerinspects site before storing commodity 66 Prophylacticinsecticide applied 100 Generalsurface application 83 Crack and crevice 50 Weedscontrolled- to reduceinsect harborage 67 Piles leveled- to facilitate future actions 83 Monitoring Scouting- variablewith seasonsand sites 100 Visualinspection - from catwalks 100 Usedgrain probe pit fall traps 76 Usedpheromones or stickytraps JJ Usedthresholds for action 100 Suppression Appliedinsecticide on placementinto storage 0 "Hot spots" handremoved as discovered t7 Top dresstreatment sprayed on pile 100 Head spacetreatment - mist or fog 67 Fumigated- selectedfacilities one or more times in past five years 83 All shellersindicated that they usedthe storagequality assurancemeasures dweloped by a USDA peanutindustry advisorycommittee @utts et al. 2OO2).These Good Management Practices(Gl"F) outlined proceduresand compliancestandards for all aspectsof receiving storing, and handlingin-shell and shelledpeanut products. Both the mandatoryHACCP plans and the voluntary GMP proceduresplaced heavy emphasis on facility managementand prwentive actionsto control threatsfrom pest infestations. AvoidanceStrategies. Avoidancestrategies were implementedby all firms. Prior to harvest,special attention was devotedto cleaningall receivingpoints, augersand conveyance dwices, trailers, dryers,and centralstoragefacilitiesto reduceinsect re-infestations. Intensive pre-storagesanitation procedures focused on eliminatingall extraneouspeanut kernels, hulls, plant debris,and eggs. The purpose was to removeharborage for pestsfrom the previou$season that could re-infestthe new crop. Mechanicalremoval methods included brushing sweeping, handpicking, compressedair, vacuumhoses, and high pressure/highvolume wash downs with 288 wateq includingthe use offire hosesby one firm. Thesemethods were usedto dislodgeinseot webbhg and residualpeanut material from rafters,wallg flooring, and equipment. Managers stressedthat pest infestationsvaried among warehousesdepending on geographical locatiorl physical condition or age of the facility, those with more southern exposuresor intense solar heating, and other factors. At two-thirds ofthe storagesites, a managerpersonally inspected each warehouse to insure completesanitation and removalof foreign matter before new peanutswere stored. A secondcomponent ofinsect avoidancepractices involved the prophylacticapplication ofa pre-storageinsecticide in andaround storage facilities. Both generalsurface and crack and crevice treatmentswere applied before peanutswere placed in storageto reduce adult and immature stagesof insecls. Surfacetreatments were applied with company-ownedspray equipmentunder the supewisionof anemployee who helda non-commercialpesticide applicator certffication.Pre-storage general surface insecticides (Table 2) were appliedliberally to all internalsurfaces, including the lower roofline, rafters,walls, and the outsideperimeter of storage buildingsto furtherreduce potential re-infestations. Generalsurface applications were appliedtwo weeksor more in advanceof peanut storage.Major changesin insecticideuse were noted between 1998 and 2003. In 1998,83% ofthe firms appliedmalathion (4 O-dimethylphosphorodithioate ofdimethyl mercaptosuccine) as a generalsurface residual spray inside warehouses. In 1998,some managers were awareof Indianmealmoth, red flour beetle,and almond moth resistenceto malathion,which had been documentedby Arthur (1989). FurtheEin 1998many operators were interestedin alternative treatmentsbut expressedconcern about firture costssince continued registration ofmalathion for storedproducts was questionable.By 2003shellers had switched to otherproducts when' malathionwas no longer registeredfor peanutsbut was still availablefor storedcereal grains. In 1998, one-third of the firms were applying cyfluthrin [cyano(4-fluoro-3- pheno:ryphenyl)methyl3-(2,2-dicholorahanyl)-2,2- dimethylcyclopropanecarborylate,las a general surface treatment at one or more sites. This synthetic pyrethroid, commonly the "Tempo" formulationfor storagefacilities, was more costlythan malathionbut was particularly useful where severepest outbreaksoccurred in the past. Between 1998 and 2003 nearly all operatorsroutinely adoptedcyfluthrin as a pre-storagetreatment. This chemicaldemonstrated good efficacyagainst several pests ofstored peanutswhen applieddirectly to peanuts(Arthur 1995). Insecticidalproperties of diatomaceousearth @E) havebeen well known for decades. But in 1998there were no reports ofthis product beingused at any peanutstorage sites in the southwesternU.S., perhapsdue to the ready availability and efficacy of other insecticides. Howwer, by 2003 at leasttwo ofthe six warehouseoperators were exploringthe useofDE as a generalsurface treatment in oneor morestorage units. DE was appliedto walls andother surfacesas a pre-storagegeneral surface treatment, with reportedlyadequate protection. Use ofDE tendedto be morecommon in the centraland westem areas of Texasand Oklahoma and easternNew Mexico wherelow relativehumidity prevailed. There were no reportsofDE being usedin southernareas (south ofsan Antonio, Texas)where stored insect problems tend to occur earlierand are more se't/€re.Synergized pyrethrins (natural pyrethrins plus piperonylbutodde) were labeledfor pre-storagegeneral surface treatment but respondentselected not to usethis product due to high costs. Crack andcrevice treatments were used by halfofthe managersprior to placingpeanuts into storage.Insecticidal treatments were appliedby handsprayer to smallcracks and fissures in storagefacilities where adult insects or eggsmight be harbored.In 1998,bendiocarb [2,2- dimethyl-1,3-benzodioxol-4-ylmethylcambamatel was used by 50%ofthe managersin rotation with other chemicalsin resistencemanagement progftms. However,this carbamatewas not re- registeredin 2000 afterFQPA reviews. In the 2003 survey,there was no mentionofthe product 289 or any lamentof its regulatorydemise. In 1998one-half of the managerswere using ryfluthrin asa crack andcrevice treatment to augmentthe generalsurface applications of malathion. Five yearslater, when malathion was no longeravailable, with the exceptionof somesites with severe insectproblems, crack and creviceuse of cyfluthrin nearlyceased since it was commonlybeing usedas a generalsurface application. TABLE 2. Insecticideand Fumigant Used in PeanutStorage in 1998and 2003 in the Sorttt*este* U. S Extentofuse Treatment (% of firms) Commentson usepatterns or changes andpesticide 1998 2003 Pre-storage:surface malathion 830 Use ceasedwith insectresistance and non re-registration cyfluthrin 33 83 Useincreased as O.P. use phased out diatomaceousearth 033 Finelyground silica oxide sprayed on walls Pre-storage:crevice bendiocarb 500 Discontinuedin 2000 with carbamate withdrawals cyfluthrin 50 17 Creviceuse decreasedas surfaceuse increased In-storage:top dress malathion 100 0 Sprayedon pile surface,based on scouting pyrethrins r7 50 Sprayedon pile surfaceat frequent intervals diatomaceousearth 050 Applied prior to insectbuild up; dusty, costly In-storage:head space dichlorvos 33 33 Appliedby timedfoggers resmethrin t7 67 Foggedor mist sprayedas neededfor adults pyrethrins t7 50 Spraydirected toward ceilings for adults Fumigation phosphinegas 83 83 Costly,facility sealed; used as a last resort Extemal areasaround warehouses were periodically mowed to reduceweedy vegetation that might provide harboragefor insectsand rodents. Somesites applied glyphosate [N- (phosphonomethy)glycine]to kill weedsto reduceharborage and fire hazardsbut no use of residualherbicides was reported. Additionally, one-third of the managers appliedcyfluthrin aroundperimeters and exteriorwalls to suppressinsect numbers and reduce migrationinto facilities. Monitoring. All firms had well-establishedroutines for monitoring insectpopulations while peanutswere in storagebetween Septemberand July. Scoutingwas conduc{edmore frequentlyas the lengthofstorage and ambient temperatures increased. These findings were confirmedin a USDA survey(Anon. 2001). For example,in the fall monthsthrough January, pilesin all facilitieswere checkedonce a monthsince storage conditions were relatively cool and 290 insectswere lessactive. Someregional differences were noted in frequenoyof inspection. In central and south Texas,inspections increased to twice a month by Februaryand March . As ambienttemperatures continued to inoreasein the springand summer, monitoring was increased to weeklyinspections and later to twice a week or morefrequently, depending on prior findings. Scouting in Oklahom4 the Texas High Plainq and easternNew Mqrico followed similar frequenciesbut was initiated later sinceseasonal temperatures and lower humidity delayedpest developmentin thesemore northern regions or higherelevations' In 83oloofthe warehouses,coned piles ofpeanuts were leveledat the time ofstoragefor improvedflow of forced ambientair, betterinspectio4 andmore uniform insecticidetreatment later. Each firm scoutedfor insectsbut employedslightly different proceduresin checkingfor wingedadutts and crawling larvae. Action thresholds varied, based upon seasonal and looation factors,prior histories,and storage structural features. Inspections were initiated at specificsites where early outbreakshad occurredin the past. Outbreakstended to occur first nearthe tops ofpiles or wheredirt or frneparticles had accumulated during storage. Employees scouted from "flyers" "millers" cat-walksin warehousesby looking for or (winged adults)above the peanut pile. Perimeterareas near the floor were q(aminedfor Indianmealmoth larvaein loose shelled kernelsand warehouseswere checkedfor lanae crawling on walls. AJleast67Yo ofthe operatorsused perforated grain probe pitfall traps to monitorpests. Pitfall traps, consistingof 45 cmJong (18 inch) plastictubes with perforationsalong the side were insertedvertically in the upper surfaceofthe pile at five to 20 locationsin eachwarehouse for later qramination. Insectscrawling through the surfacelayer of peanutsdropped through perforations into the tube and were easily observedwhen traps were inspected. An action threshold of five insectsper tube was mentionedbut chemicalintervention varied with the seasonaloutlook for additionalpest emergenceand storagetemperatures. At leastone-third ofthe firmsreported using pheromones or glueboards to monitoradult insectpopulations. Lures were placed in hangingtraps with a sticky surfaceor in perforatedtube traps. Sticky traps were placedat 3 to 5 m distancesalong cat-walks to monitor for adults. One managerreportedthat quality assurance representatives from one food manufacturerperiodically worepheromone impregnated badges during site inspections to checkfor insects.Another firm indicatedthat they discontinuedusing pheromonesto avoid attractinginsects into the faoility. Suppressionand Control. There were no reports ofappllng protective insecticides when peanutswere being conveyedinto storagein the southwesternU.S. Chemicalcontrol methodswere implementedonce action tkeshold numberswere reached. However, tactics varieddepending on seasonalconsiderations and pest populations.For example,in the fall and the onsetofcooler weatherthe presenceofinsects was ofless concern,in contrastto insects foundin the springwhen populations could increase rapidly as ambient temperatures increased. Severalstrategies reported by firms are summarizedbelow. Identification and removalof localizedinfestations were important strategiesin the fall in two operations."Hot spots" were hand-sackedto removeinsect-infested spots to avoid premature use of chemicalsand were followed by more frequentmonitoring. In the spring, chemicalapplications were deferreduntil company-definedeconomic thresholds were reached, as confrmed by monitoringprograms. Chemicalcontrol was initiated soonerin southernareas in Texasthan in northernand western regions of the southwesternU.S. Onceinsects were observedat an action level, top dressapplications, head space tr€atments, and/or fumigation were initiated. In-storageinsecticides and use patterns oommonly practiced in the southwestern peanutindustry are summarizedin Table2. Top Dress Trealments.Top dressinsecticides (treatments placed over the top and exposedsurface of piles) were sprayedby workers from catwalksipside warehouses. These treatmentsprotected the upper I 5 cm surfacelayer where lawae were mostprevalent. Managers preferredto applyinsecticides to leveledsurfaces rather than coned piles. In 1998,malathion 291 was the top dresstreatment of choicesince it was economical,easy to apply, and appliedonly wheninsect populations were noted in the late springor summer. By 2003,top dressuse of malathionhad totally disappeareddue to lack of re-registration.During this five yearperiod use of syngergizedpyrethrins increased from 17 to 50yo. However, the most 4otablechange was in the trial useand partial adoptionof DE asa top dresstreatment. Therewas no mentionofDE in 1998but by 2003, 50% ofthe managerswere experimentingwith this inert insecticide.The productwas being used in a limitednumber of warehouses,particularly those with a historyof late seasoninsect problems and in drier westernareas. Operatorsindicated that they were still evaluatingthe overallefficacy and protection offered by DE andplanned to experimentat several sites. Managersexpressed concern over the four to five fold increasein the cost of DE, comparedto previousoptions, the increaseddust from DE in thewarehouse, and dustiness inside the shellingplant. Mist and Fog Trealments. Head spacetreatments were usedto control winged adults and preventinfestation outbreaks. Applications were based on the volumeofair spacebetween the surfaceofthe peanutpile androofline andwere generally administered from pressurized canistersattached to automatedfoggrng and misting devices mounted inside warehouses. Dispenserswere timed to applyan insecticideduring non-working hours, most commonly on alternateevenings. Dictrlorvos (2,2-dictrlorovinyl dimethyl phosphate) was used in at leastone- third of the operations.Managers did not expressconcern about potential loss of this OP insecticide.Impregnated strips containing dichlorvos had not beenused in the pastfive years. Resmethrin ([5-phenylmethyl)-3-furanyl]methyl2,2-dimethyl-3-(2-methyl- 1- propenyl)cyclopropanecarborylate),nominally used in earlieryears, was being administered in two-thirds of the operationsin 2003. Use was particularlyimportant in protectingshelled peanuts.Use of synergizedpyrethrins in headspace increased only nominallybetween 1998 and 2003due to cost considerations. Fumigation. Managersconsidered flrmigation as a treatmentof last resort after insect populationswere no longer suppressedby top dressor headspace treatments, and further outbreaksposed seriouseconomio threats due to increasedtemperatures and additional generationsof insects.Fumigation was practiced in a limited numberof storagefacilities by 83% of the firms in late springand summernear the end of the shellingperiod to control late season outbreaksofinsects. Managersindicated that fumigationwas costly,time consuming,imposed downtime, limited worker re-entry,and did not provideresidual control. Aluminumphosphide was the only fumigantavailable for shellersand was appliedby warehouseemployees who held a non-commercialpesticide applicator license. Phosphine gas, liberatedwhen pellets or tabletswere exposed to atmosphericmoisture, was applied using site- specificfumigation management plans. Theseplans contained provisions on sealingthe facility to retain phosphidegas, protective clothing and other safetyprecautions for workers, placard notices,and building security to preventre-entry. After application,phosphine concentrations were periodicallymonitored using hand-heldcolorimetric equipment. Industrial hygienelwels were checkedbefore warning placards and security locks were removed.There was no mention of anyexposure problems for employeesor communitieswhere storage facilities were located. Insecticide [Jsage. The amountof the southwesternpeanut crop treated with an in- storageinsecticide varied fiom l0 to 40Yoeach year, depending on lengthofstorage, seasonal progressin shelling shipments,geographical location, and local environmental conditions. While the surveydid not seekconfidential information on specifictonnages treated, from commentsby severalmanagers we estimatedthat an averageof 18 % of the total crop was treatedwith one or more in-storagechemicals. It was notedthat the actualtreated product was typically the top layerofin-shell peanuts,where the raw kernelwas protectedby the hull andwas not directly exposedto an insecticide. 292 Shellersexpressed concern on continuedavailability ofcurrentlyJabeled pesticides and prospectsfor new products.While cerealgrains provide sufficientlylarge markets for economic rcturns for pesticideregistrants, the peanutstorage market is relatively small. Consequently, shellerso TABLE 3. VertebratePests and Control Tactics in StoredPeanuts in the SouthwesternU.S. Pestsand Tactics o/oof firms reoortine Pests Feralpigeons 100 Englishsparows 33 Ratsand mice 67 Tactics Restrictivenetting and exclusions 83 Mowing or herbicides-for habitatmodification 67 Warfarin baits - in feed or water 50 Electric fencing 33 The Norway rat (Rattus norvegicusBerkenhout) and, to a lesserextent, the roof rat (Ranus rattus Linnaevs) and housemouse (Mlrs musctrlusLinnaeus) cause losses in stored 293 peanutsdue to fecalcontarnination. These rodents are excellent climbers, have high reproductive capacities,vector severalhuman diseases, and contaminateten times more seedthan they consume(Ilygnstrom and VerCauteren1995). Shellersmentioned that rat droppingswere difficult to screenfrom peanuts,were not appreciatedby food processors,and were highly undesirablein food products. Sincethese vermin requirefood, water,and harborage, all of which are readily availablearound peanut storage facilities, preventivecontrol measureswere practiced.Externally, mowing, sanitation, rodent-proofconstruction, and netting were cornmon prwentivemeasures, One firm usedelectric fencing with positiveand negative wires placed 2.5 to 4 cm abovepaved surfaces to deterrodents. Populationcontrol measures were employed insidewarehouses. Rodenticides and trapswere strategicallyplaced near feeding sitesand runways.Warfarin [3-a-acetonybenzyl)-4-hydrorycoumarin], ananticoagulant, was administered in food blocksor water baitsat one-halfof the sites. Waterbait stationswere deemedto be particularlyeffective for rats in summer.Pellet formulations of warfarinwere not used,to avoid physicalcontamination with similarly-sizedpeanut kemels. Entrapments included snap traps, glue boards,and multiple catch boxes,with and without baits. Feral housecats (Felis domesticusLinneaus),present at somesites, helped to suppressrodent populations. ACKNOWLEDGMENT Cooperationby membersof the SouthwesternPeanut Shellers Association made this studypossible. Max Grice ofBirdsong Peanutat Gorman,Texas, and FrankArthur at the Grain Marketing andProduction Center, USDA-ARS , Manhattan,KS offerednumerous suggestions duringthe courseofthis work. Roy Parker,M.O. Way,Steve Brown, andRiley Curb provided helpfulreviews and comments at variousstages of preparationof the manuscript. LITERATURE CITED Anon. 200I . Agricultural chemicalusage postharvest applications - peanuts,rice, andsorghum. NationalAgric. Stat.Serv. Ag Ch l(01). lp. Arthur,F. H. 1989.Effects of cleaningpeanuts on insectdamage, insect population growth and insecticidalefficacy. Peanut Sci. l6: 100-105. Arthur, F. H. 1995. Susceptibilityof fifth-instar Indianmealmoth and almond moth (Lepidoptera:Pyralidae)tocyfluthrinresidesonpeanuts.J.Entomol.Sci.30:318-323. Arthur,F. H., andS. L. Brown. 1994.Evaluation ofdiatomaceous earth (Insecto) andBacillus thuringiensisformulation for insectcontrol in storedpeanuts. J. Entomol. Sci. 29:176- 182. Brower,I. H. 1983. Utilizationof stored-productLepidoptera eggs as host by Trichogramma pretiowmNlev (tlymenoptera:Trichogrammatidae). J.Kansas Entomol. Soc. 56: 50-54. Butts,C., R. Crozier,J. Smith,andB. Willingham.2002.Good agricultural practices forfarmer stockstorage and handling. Chapter 3. 1lr R. Henningand D. Cowart(ed.) Amer.Peanut CouncilGood Management Practices Handboolg Alexandria, VA. Coble,H. D. 1998. A new tool for measuringthe resilienceof IPM systems:the PAMS diversityindex, pp.l52-154. In E. Day, (ed.)IPM MeasurementSystems Workshop Proceedings.Am. FarmlandTrust Ctr. for Agr, in the Environ.Chicago, IL. Corral,Wong F. J.,M. O. CortezRocha, I. BorboaFlories, and F. BustamanteAndrade. 1992. Insectspecies infesting grain storedin rural communitiesin the northeastof Sonora, Mexico. SouthwesternEntomol.. 17 :327 -33 l. Hagstrum,D., andP. Flinn. 1995. IPM in grainstorage and bulk commodities,pp.2Ol-205. In Krischifi,V., G. Cuperus,and D. Galliart (ed.) Stored Product Management. OklahomaAgric, E>rt.Serv. Cir. E-912. Hygnstrom,S. E., andK. C. VerCauteren.1995. Vertebrate pest management in grainstorage facilities.pp.227-238. In KriscNf,V., G. Cuperus,and D. Galliart(ed.) Stored Product 294 Management.Oklahoma Agric. Ext. Serv'Cir. E-912' oflndianmealmoth Kinsinger,-andR.-A., W. H. McGaughey,and E. B. Dicke. 1980.Susceptibility almondmoth to ei[ht Bacitlusthuringiensis isolates (Leptidoptera:Pyralidae). J. KansasEnt. Soc.53 :495-500. 1995. Storedgrain management Noyes,' ' R. T., R. Weinzierl,G. w. cuperus,and D. E. Maier. techniques,pp.7l-84. 1nKrischif, V., G. Cyperus,and D. Gallant(ed.) Stored Product Management.Oklahoma Agric. Ext' Serv.Cir. E-912' Redlinger,L,-M. andR. Davis. 1982. Insectcontrol in postharvestpeanuts, pp. 520-570.In H. E. P"tt." andC. T. Young(ed.) Peanut Science and Technology' smith, D. T., M. C. Black, w. I. Grichar,and A. L Jaks. 2000. Economicassessment and fungicideuse on peanutseed in the southwesternUnited States.Peanut Sci. 27:39-44. smith, D. T., M. J. New, andJ. T. Criswell. l9j& Peanutpest management practices and chemicaluse - a surveyofthe southwestindustry. 1998Proc. Am. Peanut.Res. Educ' Soc.30:52. 295 VOL.29 NO.4 SOUTHWESTERNENTOMOLOGIST DEC.2004 SCIENTIFICNOTE THE BEE FLYI FXOPROSOPAFASCIATA MACQUART IN CENTRAL TEXAS WTTI{ NOTES ON CONSPECIFICSOCCI.JRRING IN TIIE STATE StephenW. Tabel and Scott B. Fleenof While conductinga biodiversity surveyin The Lost Pinqr forest of east-centralTexas, we collected several bee fly specimensthat we could not identify with available keys (Taber andFleenor 2003, photo p. 73). Communicationswith exp€rtsand e:omination of materialobtained from severalmuseums, including one specimenlabelled as a homotype, now indicate that the difficult taxon is actually ExoprosopaJascian Ivlacquart 1840. According to Evenhuis and GreatheadQ003), this specieshas not been reported from Texas. The confusionwas causedby errors in two identification keys affecting not only the identification of specieswithin the genusExoprosopa Ivlacquart (the most specioseof all bombyliid genera) but even the identification of the genus itself. These same problemsare likely to provide obstaclesto otherg and our purpos€here is to point out the mistalresthd we encounteredand where possibleto rectiS them. The materialconsisted of three adult males collected in Bastrop Stde Parlg Bastrop County, Texas. Two were collected on 17 September1995, one on 4 October2003. The latter was capturedwhile nectaring at field sruke-cotto4 Froelicka Jlorifuu, in a meadow of little bluestem grass,Sckrelryr'hm &oryriun. Using the most rec€nt key to Nearctic bee fly genera (tlatl l98l), we correctly identified the genusas Exogarya. This was supportedby the wing venation pattern as illustrated by lfull (1973). However, using the key in Curran (1934), the genuscould not be correctly determineddue to the sameerror that causedus to later misidentiS the flies with the sameautho/s key to F^rqrorcpa species(Cfrnan 1930). The problem is the length of the proboscis. Neal Evenhuis of llawaii's Bishop IVfuseumexplained to us via email that the probosoisleqgth varies so much due to its ability to r€tract and extend that it is risky to use it as a oharacter. When insertedinto long corollasthis structire may extendup to twice its retractedlength (tluefler 1966). In our own er lOiperar Bombyliidae 2Aiobgy Dqrarfinent, Saginaw Valley StCe University, 7400 Bay Road, University Center,Michigan 48710 3rcs W fstn SftrId.#2f/.,Austin, Toras 78705-1443 297 secondcouplet (p.2) One using thesekeys may go far astray,for none ofour specimens had so slrort " plobor"ir. The-original E. lasciata descrip,tionalso contradictstlre keys with its commentthat the proboscisprojects from the face by the length of the animal's head(Macquart l8aQ p. 5l). We setttedthe identification of tle speciesat hand by comparingthem to a labelled homotypeand severalother specimensall identified by R. H. Paint€r Paint€r examined fragmints of what he believed were E. fasciata type specime,nsin the Paris Muszum of the antenoa @inter and Painter 1962). We comparedthe habitug the third segnenJ and its stylus, and especially the male genitalia which we dissectedfrom one of our specimensaoi from one ortire nies iaeotined as E. larciataby R. H. Painter- very few oithe old speciesdescriptions offer theseillustrations that we now believe to be crucial, and male g"nitntio had nwer before been illustrated fot E. fascian The genital capsule of one of-our Texas specimensis illustrated here (Fig. 1). It appearsto b-eidentical in every way to that ofthe Painter specimenand immediatelyseparable from illustrations of all oitherspecies that we have seen(Johnson and Johnson1958, Ifull 1973). Difrerences betweenpiobosoides and antennaeof this and other species,wch os Exoposopa sordifu Loeur, ari not neady so obvious. The shapeofthe epiphallusin particular showspromise as a meansofseparating one taxon from another. FIG. l. Exoprosopfasciaa, male genitalia; left lateralview. Fifteen Ercprosopbee fly speciesare reported from Tqras accordingto the Worl,d catalog of Bombyliidae @venhuisand Greath€ad2003). Thesearc E. agassizilaew, E. albicotlorts Painter, E ownala Painter, E decora Loew, E dvlsa (Coquillett), 4. dorcadionOstenSackeqE.dorisOstensacken, Efascipemis(Say),,8 lruIliPdntq,E. E ingezs Cresson, E. iota Osten Sacken" E. meigenii (Wiedemann), ^pueblensis Jainnicke, E. rhea OstenSaoken" tnd E. teruu Curran.With the addition of E fasciata as reportedhere and an overlookedrecord for E sordidn (seeCole I 969, p. 254), the totd rises-toseventeen. Additional collecting and examinationof museummaterial is likely to increasethis number. Six Toras t2:rawef,e available for illustration (Fig. 2). We would like to see two additional coutributiors to the problem of Etoprorya idemification' First, more use of male genitalia when describing new speciesand less regard paid to proboscis length. Second an illustrated key to all Texas speciesofthe genus- This would save Jtot of time and trouble for those of us who are engagedin biodiversity surveys. We ttant Neal Evenlnris of the Bishop lvtuseurn,Edward Riley of Texas A&M University, and Lars Vilhelmsen of The University of Copenrhagenfor loaning material essentialto the sohrtionofthis problem, as well as David Riskind of The TexasPar*s and Wildlife De,partmentfor is*ring Scientific StudyPermit No. 2l-01 and earlier versionsof the same. 298 FIG. 2. Six of 17 Eroprosqa speciesknown to ooqrr in To LITERATT'RE CITED Cole F. R., and E. I. Schlinger. 1969. The flies of westemNorth America Univ. Calif. Press,Be*el€,y, CA Cuna4 C. Il 1930. New Diptera from North and Ccntral America. Amer, Itfus. Novit. No.315. Curran, C. H. 1934. The families and gcnera of North American Dipt€ra. Tlp Ballou Press,New Yort Evenlnriq N. L., ard D. J. Greathead. 2A08. World catalog of bee flies @iptera: Bombyliidae) web site. [htp:/lrbs.bishoprmrseum.org/bombcat{. I{rll, J. C. 1981. Bombyliidae, pp. 589-602. fh [Iarual of Nearctic Dipter4 Vol. l. J. F. McAlpine et al. "coordinators.n ResearchBranc\ Agric. Csnadq Monograph No.27, CanadianGov. Pub. Cemre,Ifull Qrebec, Canada. Huether, C. A., Jr. 1966. The ortent of genetic \xuiability for a canalizedchamcter corolla lobe numberin naturd populationsof Litwtthts @enth ). Ph.D. dissertation, University of California" Davis. Hull, F. M 1973. Bee flies ofthe World. SmithsonianIDstinrtion Press, Washingo4 D. c. Johnson,D. E., and L. M. Johnson. 1958, Nes, and inzufficiently lmwm Exoposqa from the far west @ip,tera:Bombyliidoe). Gr€atBasin Nat. 18: 69-84. Ivlacquart, J" 1840. DiptArer Exotiques: Nouveaux ou peu connus. M6m. Soc. Sci. Agric. Lille 1840:t-135 + 2l plates. Painter, R H., and E. M Painter. 1962. Notes and rcdescripions of types of North American Bombyliidae (Diptera) in Europeanrmneums. J. Kansas.Ent. Soc. 35: 2- 164. Taber, S. W., and S. B. Fleenor. 2003. Insectr of the Toras Lost Pines. Texas A&M University Press,C;ollege Station, Texas. 299 vol.29 NO.4 SOUTHWESTERN ENTOMOLOGIST DEC.2004 SCIENTIFICNOTE BIOCHEMICAL COMPARISONOF FIELD AND LABORATORY POPULATIONS OF PODISUSMACULIYENTNS (HETEROPTERA:PENTATOMIDAE)IN FORIDA' JesusaCrisostomo Legaspi2, Jeffrey P. Shapiro3,and Benjamin C. Legaspi,Jr.a Podisusmaculiventris (Say) (Heteroptera: Pentatomidae) is an importantgeneralist predator commonly found in grape vineyards and natural vegetation in north Florida. Beginning in May 2002, P. maculiventris were collected from the field using nine clear covered pheromonetraps placed in a muscadinegrape vineyard at the Florida A&M UniversityCent€r for Viticultwe in Tallahassee,Florida. Samples were collected daily except the week-ends.The trapscontained a pheromonemixture (Aldrich 1988),and a vial of water with a cotton wick. Individuals of similar ageQ0 malesand 20 females)were randomly collectedand weighed from a laboratorycolony fed Tenebriomolitor L. (coleoptera: Tenebrionidae). In preparationfor biochemicalanalysis, insects were homogenizedon ice using a Polyhon homogenizer@rinkman). Protein was determinedusing the bicinchoninic acid (BCA) assay(Pierce) with bovine serumalbumen standards, Values were interpolatedfrom the standardcurve, and resulting values(pglpl) were convertedto pglinsect.To determine yolk proteincontent by ELISA, absorbanceswere compared in an indirect antigenELISA to standardhomogenates of eggs from P. maculiventris (Shapiro and Ferkovich 2002). Aqueousextracts of whole insectswere subjectedto lipid extractionwith chloroform and methanol(Bligh andDyer 1959),One milliliter of wholeextact wasmixed with NaCl and methanol/chloroform, vortexed and micro-cenfrifuged, The subnatantwas removed, cbloroform addedand the processrepeated. Fatty acidswere hansmethylatedprior to gas chromatography.Hexane and methanolicboron Eifluoride solution were added,and tubes werecapped and heated. After cooling,hexane and l{ro wereadded, vortexed, and the upper hexanephase was removed.This was repeated,and combinedhexane fractions were dried underNr. Sampleswere analyzed on an Agilent 6890GC. Femalesfromthelaboratorycolony(85.0mg)weresignificantlyheavierthanmales (57'0mg) (t-test,P < 0.05).Average body weight of a field-collectedfemale (56.0 mg) was also heavierthan that of the male (43.0 mg). Laboratory-rearedfemales were significantly heavierthan field-collected females. Comparisons of live bodyweights of P. maculiyentris collected in this study were made with body weights of laboratory-rearedfemales under different feeding regimens(kgaspi et d. 1996).Results indicated that the field-collected femaleshad maximal live body weights comparableto femalesfed one prey item every 9 daysin thelaboratory (Fig. l). Thesefindings are similar to thoseobtained by Legaspiet al. (1996) suggestingthat P. maculiventrisdoes not requirehigh prey numbersto survive and possiblyto reproduceunder natural conditions. tThis articlereports the resultsof researchonly. Mentionof a proprietaryproduct does not constifutean endorsenrent or recommendation for its use by USDA. ARS,CMAVE, FAMU - Centerfor BiologicalControl, Tallahassee, FL 32308, ]USOA,'USDA, ARS,CMAVE, 1600-1700 SW 23'Drive, Gainesville,Florida 32608. aEmployedby StateofFlorida, conhact through senior author. 30t t Ad l,bitum € 3'days ul 9-days +l c, E .9 0 B 0 N 0 c n6 Month(2002) FIG 1. Live body weightsof Podisusmaculiventris (mean mg + SE) collectedby pheronronetraps in Tallahassee,Florida, from May to July 2002(histogram indicates iamplesize). The solid lines indicate the mean body weights of femalesprovided prey ad libitum,or oneprey item every3 or 9 daysin the laboratory.The shadedalea denotes feedingrates corresponding to ad libitum and every 3 days(Legaspi et al' 1996). To investigategeneral fitness and reproductivecapacity in field vs. colony-reared females,contents of protein and fatty acids in femaleswere analyzed.Total solubleprotein washighest in femali colony-rearedinsects (Table l). This generallyreflects the larger size of the colony-rearedfemales, and more specificallytheir accumulationof nutrientsand incorporationinto developingeggs. Colony-reared females contained 37%o more soluble protein (9.46 vs. 6.90 mg/insect)than field-collectedfemales. Colony-reared females = = containedslightly more yolk proteinthan field-capturedfemales (t 2.7; df 10.7;P < 0.05).Percentage contents ofthe methylester derivatives ofthe six mostprevalent fatty acids werecomparedlncolony-rearedandfield-collected females (Table 2). Foru fatty acidmethyl esters(FAME) derivativessignificantly differedin percentageoftotal betweencolony-reared andfield-collected insects. Pircentages ofthe methylesters ofpalmitic acid(16:0) and oleic acid (18:l) were significantlyless in field-collectedfemales than in laboratory-reared femalls. while those of stearic (18:0) and linolenic (18:3) acids were greater in fi eld-collectedfemales. with the markedly higher levels of protein in colony-rearedfemales, reduced reproductivecapacity might be expected.Yolk proteindeterminations, however, indicate that field-collectediemales contained levels of yolk proteinonly slightly lower than thosein colony-rearedfemales (Table I ), suggestingthat field-reared females are able to successfully ,"produ." despite low prey intake. Shapiroet al. (2000) demonstratedthat yolk protein synthesisand appearancein hemolymphof Podisusfollows feeding,and that the quality of 302 TABLE l. Total Body Protein (+ SE) in FemalePodisus maculivenffis (Field Versus Solubleprotein (mg/insect) N Yolk Protein (rrglinsect) N Field 6.90+ 0.52 23 84.90*15.86 l1 Colony 9.46*0.95 9 98.82+37.96 5 TABLE 2. PercentageContents of the Methyl EsterDerivatives of the Six Most Prevalent Fatty Acids (FAME) in Colony-RearedVersus Field-CollectedPodisus maculiventris Females Field __u9!9!L- SD l6:0 20.74 7.47 31.r7 2.65 l6:I 4.68 3.44 4.66 4.01 18:0 10.44 2.45 6.22 2.5 5 l8:I 30.57 6.46 42.29 4.77 18:2 t7.07 6.87 13.31 3.03 l8:3 16,50 6.t9 2.35 0.73 Total 100.00 100.00 diet affects the onset of appearance.Present results therefore indicate that field-collected femaleslikely fed on prey sometimeprior to collection.Further biochemical comparison betweenfield and laboratorypopulations may provide a basisfor estimatingthe impact of predatorson populationsof their prey, and conversely,the effect of prey on survival and reproductivefitness of predators. We thankI. Baez(USDA, ARS), J. Head,A. Donnell,and W, Allen (FAMU) for technicalassistance. Helpful reviews wereprovided by J. Petersand M. Haseeb(FAMU). LITERATURECITED Aldrictr,J. R. 1938.Chemisty andbiological activiryofpentatomid sex pheromones. Pages 417-431.InH. G. Cutler[ed.] Biologicallyactive natural products: potential use in agriculture.American Chemical Society, Washington, D.C. Bligh,E. G., andW. J. Dyer. 1959.A rapidmethod of total lipid extractionand purification. Can.J. Biochem.Physiol. 37 : 9ll-917. Legaspi,J. C., R. J. O'Neil, andB. C. Legaspi,Jr. 1996.Trade-offs in bodyweights, egg loads and fat reservesof field-collectedPodisus maculiventris(Heteroptera: Pentatomidae).Environ. Entomol. 25 : | 55 -l 64. Shapiro,J. P., H. A. Wasserman,P. D. Greany,and J. L. Nation' 2000. Vitellin and vitellogeninin thesoldier bug , Podisusmaculiventris:ldentification with monoclonal antibodiesand reproductiveresponse to diet. Arch. Insect Biochem' Physiol. 44:130-l 35. Shapiro,J. P., and S. M. Ferkovich.2002. Yolk proteinimmunoassays (YP-ELISA) to assess diet and reproductive quality of mass-rearedOrius insidiosus (Heteroptera: Anthocoridae).J. Econ.Entomol. 95: 927-935. 303 vol.29 NO.4 SOUTHWESTERNENTOMOLOGIST DEC.2004 SCIENTIFICNOTE THE GENUS ME CIDEA (HETEROPTERA:PENTATOMIDAE) IN NEW MEXICO C. S. Bundy Departrnentof Entomology,Plant Pathology,and Weed Science, New Mexico StateUniversity Las Cruces,NM 88003 The stink bug genusMecidea Dallas is representedin North America by two species,i4 major sailer md M. ntnor Ruckes. Taxonomically, these two sirecies are the only continental North American members of the tribe Mecideini within the subfamily Pentatominae.Originally (Uhler 1876),the continentalfauna was consideredto consistof a single species,misidentified as M. longula stal (a speciesnow known only from the west Indies fSailer 1952]). Subsequently,continental material previously treated as "M. Iongula" was redescribedby Ruckes(1946) as M. minor, andby Sailer(1952) as M. major. Little is known about the biology and seasonaldevelopment of these insects. They are gorerally consideredto be grass-feedersin xdric and semi-xericenvironments lSailer ilSZ; *A n"* potentialto be grasspests @uckes 1938). Both specieshave been reported from side-oats gruna, Bouteloua cartipendula (Michaux) Torr.; bermudagrass,cynodon dactyton (L.); Lehmarurlovegrass, Eragrostis lehmannianaNees; and spinach,Spinacia oleracea L. II addition, M. rnajor has been collected from wild oat, Avena fatua L; bamyardgrass, E-chinochloacrus-galli (L.) Beauv.; bush muhly, Muhrenberyiaporteri scribn.;-sorfium, sorghum halapense(L.) Persoon;wheil., Triticum aestivumL.:,Biomus sp.;senecio sp.; and cotton, Gossypiumhirsutum L.; whereas,M. minor has been collected from black grama, Boutelouaeriopoda (Torr.) Torr.; aridland goosegrass,Chenopodiun desiccatum A. Nelson (as c. pratericola desiccatum [A. Nelson] Aellen); Bothiochloa sp.; Digitaria sp.; Eragrostissp.; andsporobolus sp. (Jones1993, Mcpherson 19g2, sailer 1952,Thomas and Werner1981, Watts 1963). Mecidea minor rmges from Mexico and Califomia north to South Dakota and east to Texas. Mecideamaiorhas a more eastemdistribution, and has beenreported from Arizona and Texas north to Kansasand Missouri and east to Illinois (Froeschner1988). In New Mexico, only M. minor has been repodedpreviously 1946,sailer lO52). The *M. @uckes genus.originally was reported from the state (as longala') by Uhler (1g76), from material collected on an expedition in 1873. In his original descriptionof M. maigi, Sailer (1952)reported no occurrencesof this speciesin New Mexico. D-*i"g the springo,f zooz, oneM. maior femalewas collected from common oat,Avena sativaL, The identification of M. major was confirmed in New Mexico by the author the following year using males, promptinga closerexamination of Mecideain New Mexico. During 2003, various plant species,primarily grasses,were surveyedin southemNew Mexico for presenceof both speciesof Mecidea. From April to Novernber2003, sweep sampleswere taken every I to 2 weeks,primarily from the south centralportion of the state (rear Las cruces), but also fiom the eastem(Bitter Lake National wildlife Refuge, chavez co.) and southwestem(Gray Ranch,Hidalgo co.) regionsof New Mexico; a total of = 20 field sites were visited during the survey. Sweepswere taken from plants growing in 305 patcheslarge enoughto obtain an unadulteratedsample. Collection datawere supplemented with label information from specimenshoused in the New Mexico State University ArthropodMuseum and Biology InsectCollection. Table I indicatesnew host recordsfor both speciesof this stink bug in New Mexico. Most plant species, including those upon which the bugs were reproducing (based on presence of immature stages and copulating adults), were grasses. They were most commonlyfound on rangegrasses. TABLE I . New Hostlecords of M. na,lor,,lndM' jzrzor New Host Records -l-ocation Date" Stageb Mecidea maior Aristidapurpurea (Wright's threeawn) DoftaAnaCo. 4June2003 NA Avenasativa (commonoat)" DoffaAna Co. 15 April 2002 NA Boutelouaeriopoda (black grama) DofraAnaCo. 4June2003 NA Boutelouagracllrs (blue gama) DoffaAna Co. 20May2003 NA Hordeum vulgare (commonbarley) DoflaAna Co. 22May 2003 NA H or deum j ubatum (foxtail b uleY) DofraAna Co. 2lMay 2003 ENA M edi cago sat iv a (alfalfa)" DoflaAna Co. April 2003 A Pleuraphisril uticd (tobosa$ass) DoffaAna Co. June-Oct.2003 NA Sporoboluscontractus (spike dropseed) DoflaAnaCo. 16June 2003 A NA Sporobolusflexaoszs (mesadropseed) ChavesCo. 6 August2003 Mecideaminor ,qrittidop"rp*ra (Wright'sthreeawn) DoflaAna Co' 4 June2003 NA NA Bouteloia gracilis Slue grama) Dofla Ana Co. 20May 2003 Gossypiuihirsutum (upfandcotton) DofraAna Co. August 2003 A 1982 A GutiLrreziasarothrae $room snakeweed)'d DofraAna Co. 9 September Pleuraohismutica (tobosaerass) Dofla Ana Co' June-Oct'2003 NA " Date insect was first collected on host. "E' 'N" "A" immatute o 1'l" nf" stages collected are represented by an for egg, for nymph, and for adult; identification inferred ftom associatedadults. " Host plant information is basedon a single collection. d Host olant information was taken from data label of museum specimens' Specifically,14 major reproducedon commonbarley, foxtail barley,and mesa dropseed; whereas,M. minor reproducedon Lehmann lovegrass. In addition to sideoats grama' reported previously to commonly support populations of Mecidea (Sailer 1952)' both species"iro *"r" iound to reproduceon Wright's threeawn,Aristida Purpurea Nutt. var. wrightii (Nash) Allred; blaci< grama; blue grarn, B. gracilis (Willd.ex Kunth); and tobisagrass,Pieuraphis mutica Buckl. Adults of i4 maior were collecled from common oat, alfalfa, andspike dropseedwhile adults of M. minor were reportedfrom upland cotton *d broo- snakeweed.Feeding was observedfor thethree Bouteloua species, Aristida, and Pleuraphis in the field and as part ofa laboratorystudy (cSB, unputlished data). Feeding is impliiedfor plantslisted above hosting reproducing populations of the bugs. It is unclear if ttre stink bugs fed upon the broadleafspecies. At the time M. naior was collected from alfalfa, most gr""t.r were still dormant, and the alfalfa had lush, green tissue. Therefore, the plant might have been a harborage site for Mecidea until preferred species were avaiiable.This informationis presentedhereto betterelucidate the biology of a pentatomid tribe aboutwhich little is known. 306 Voucher specimensof both speciesof Mecidea are depositedin the New Mexico State Arthropod Museum. I thank Kelly Allred (I{I\4SU) for identification of plant species,Ken Butts,USFWS manager, and Gordon Warrick, USFWS biologist, for permissionto collect in Bitter Lake National Wildlife Refuge, and Ben Brown for permissionto collect at the Gray Ranch, and David Richman (NMSU) for critical review of this manuscript. The researchwas funded by the New Mexico Agricultural ExperimentStation, Las CruoesNew Mexico. LITERATURECITED Froeschner,R. C. 1988.Family PentatomidaeLeach, 1815, The stink bugs,pp. 544-597.In T. J. Henry andR. C. Froeschner[eds.] Catalog of the Heteroptera,or true bugs,of Canadaand tlie continentalUnited States.E. J. Brill. New York. 958 pp. Jones,W. A. 1993.New host andhabitat associations for some AizonaPentatomoideaand Coreidae.Southwest. Entomol. Suppl. 16: 1-29. McPherson,J. E. 1982. The Pentatomoidea(Hemiptera) of NortheasternNorth America with Emphasis of the Fauna of Illinois. Southern Illinois University Press, Carbondale.241 pp, Ruckes, H. 1938. Additions to an annotatedlist of pentatomids (Heteroptera)of New Mexico.Bull. BrooklynEntomol. Soc. 33: 10-13. Ruckes,H. 1946.Mecidea minor, a new speciesof pentatomidfrom New Mexico. Bull. BrooklynEntomol. Soc. 4l: 86-88. sailer, R. r. 1952.A review of the stink bugs of the genusMecidea. proc. u.s. National Museum.102:471-505. Thomas,D.B.andF,G.wemer.lg8l.Grassfeedinginsectsofthewesternranges:an annotatedchecklist. Univ. ArizonaAgric. Exp. Sta.Tech. Bull. 243.50 p. Uhler, P. R. 1876. List of the Hemipteraof the region west of the MississippiRiver, includingthose collected during the Haydenexplorations of 1g73.Bull. U.S. Geol. Geogr.Surv. Territories. l: 269-361. watts, J.G. 1963. Insectsassociated with black grarnagrass, Bouteloua eriopoda, Awr, Entomol.Soc. Am. 56:374-379. 307 vol.29 NO.4 SOUTHU/ESTERNENTOMOLOGIST DEC.2004 SCIENTIFICNOTE AN ourBmAK oF oNCI DE RESp usr u LATA (coLEoprERA : GERAMByGTDAE) IN NORTHERNTAMAULIPAS, MEXICo: DAMAGE, DISTRIBUTION.AND HOST PLANTS Luis A. Rodriguez-del-Bosque InstitutoNacional de InvestigacionesForestales, Agricolas y pecuarias(INIFAP), campo ExperimentalRio Bravo.Apartado postal 172JRio Bravo, rarn, vexico sasoo The . . twig girdler, oncideres pusturata (= pusturatus)Leconte, attack several lesuminous tree speciesin southernGxas and northeastemNf.*i.;1Higr, rqs, Linsley and chemsak 1984, Rice 1989, Noguera 1993). The most preferred"hostplants are huisache_,lcacia farnesiana(L.) willd", and tepehuaje,trurorio piiiir"/enra Benth' (Rice lschlecht.) 1989). other hosts occasionally'girdlidby o. puitularo in.rua., huajillo, Acacia berlandieri Benth.; mimosa, Mimisa- lindheimerr Gray,;,"tu,nu, parkinsonia aculeata L.; ebony, Pithyll9blu.m flexicaule (Benth.) Coult.; and -.rqrir", prosopis glandulosaTorrey (High 1915,Linsley 1940,Hovore and penrose19g2, Rice l9g9). In addition,d-amage by O, pusnlata to leguminousornamental and shadetrees can be severe (rirgh fvr5, Hovoreand Penrosel9g2). Biology,behavior, and ecology of o. pustulatahas beenstudied in southernTexas,particularly ori-huisache Grieh rsrs;"LiJ.v rs+o; Hovore andPenrose 1982; Rice 1986,l9g9). In spite . of the proximity from southernTexas, there are no previousreports of a pustulala in northern Tamaulipas, across the USA-Mexico border.. Although branch girlding activity in tree legum"i ha.n" been observedin northem Tamaulipas for years, identity of the insect had not been determinedbefore. Moderate giidling activity, presumably causedby o. pustulata,was observed near Rio Bravoduring tfe rdt or tgsg. An important oulbre-af-of O. pustulara (identified by Felipe-A. Noguera from in -qNAM chamela, Jat., M6xico) occurred in northem Tamaulipas during october- November 2003.During this period,damage, distribution and host pi-ts or o. pusturata were studied in 83 inspectionsites (natural and disturbedareas) ofnorthern Tamaulipas.In eachsite, at least20 treeswere inspected. o. pustulataadults initially wereobserved during mid-September, and populations peakedfrom mid-octoberto mid-November,when branih girdling activity was intenseat levels not observedin this areabefore. Adults of o. pustiata *ir. .ornironly observed f:!ltq'^ry,rlg, and ovipositingon branches,with samehabits reported for southemTexas (High penrose 1915;Linsley 1940; Hovore and l9g2; Rice 19g6;l9g9). Atthough density was not measuredin this study,in most cases,single pairs were observedreioing ani matingon eachgirdled branch, with somebranches traruoiing murtiple pairs (up to eight). Prefened host plants by o. pusturata were huisaJhe *d t"ir"rr*:", connr-ing findings in southernTexas (Rice 19i39;.However, when both hostscoincided in mixed populations, tepehuajewas preferredover huisacire,a finding not previously reported. .{v3r1 qir{ti1g activity was also observedin tre ornamentarflamboyant, Delonix regia (Bojer) Raf.. Infrequentdamage by o. pustulata was detectedon -"rquit., ,.tama, ebony, and guamuchil,Pithecellobium dulce (Roth.)Benth. Flamboyant and iuamuchil ,"pr"r.ni new hostplant recordsfor O.pustulata. 309 I Number of trees attackedby O. pustulata in the 83 sites averaged42%o (max 90o/o) for tepehuaje,and 24Yo (max 75Yo)for huisache.Percentages of flamboyanttrees attacked in two sites were 55 and 80. However, regardlessof host plant and site, damageto the canopyvolume was usually<10%, with an averageof 4-5 (max 18) girdledbranches per tree,Nearly 90% ofthe girdledbranches broke offand fell to the gtoundor lodgedamong otherbranches. Average + STD diameterof girdledbranches was 2.86 + 0.71,2.70+ 0.37, and 2.25 + 0.32 for tepehuaje(n = 188), flamboyant(n = 35), and huisache(n = 143), respectively,similar to the reportofRice (1989)for southernTexas. Distribution of the O. pustulata outbreakin northernTamaulipas comprised an area of nearlyone million hectares,including the following municipalities:Rio Bravo,Reynosa, Matamoros,Valle Hermoso,San Fernando,Mdndez, DiazOrdaz, and Camargo(Fig. l). Severe,moderate, and low damageby O. pustulataoccurred in approximately10,40, and 50% of the area,respectively. The areamost affectedwas Rio Bravo and Reynosa.Girdling activity by O. pustulata was not detectedwest and south of Camargo into the state of Nuevo Le6n, and south of San Fernandointo centralTamaulipas. Although damageand distribution were not evaluated in the Lower Rio Grande Valley of Texas, important girdling activity by O. pustularaon tepehuajeand huisachewas also observedacross the bordernear Progreso, TX. a t'. a Nuevo Le6n Gulf of Mexico FIG. l. Distribution of O. pustulala outbreak in northern Tamaulipas,Mdxico; l= Camargo,2: Diaz Ordaz,3: R"ynotu, 4= Rio Bravo, 5: Valle Hermoso,6: Matamoros' 7-- M€ndez,8: SanFernando. October-November, 2003' 310 Factorsassociated with this O. pustulataoutbreak in northemTamaulipas remain unknown.Rainfall above average in this regionduring 2003 might havefavored survival of O. pustulata immatures, and hence abundanceof adults. In southern Texas, greater O. pustulata adult emergencewas associatedto higher humidity conditions(High 1915). Speciesriclrress and abundanceof cerambycidswere highest during the rainy seasonanrd lowest during the dry season in central Mexico (Noguera et al. 2OO2).Freeze is an important winter mortality factor for O. pustulata (Rice 1986). The last severewinter in northernTamaulipas occurred in 1989,when temperaturesdropped to -8 oC during late December.Mild winters during the last years may also be contributing to o. pustulata populationbuildup in this region. LITERATURECITED Hieh,M. M. 1915.The huisache girdler. USDA Bull. 184. Hovore, F. T. and R. L. Penrose.1982. Notes on cerambycidaeco-inhabiting girdles of Onciderespustulata LeConte (Coleoptera: Cerambycidae). Southwest. Nat.27: 23- 27. Linsley,E. G. 1940.Notes on Onciderestwig girdlers.J. Econ.Entomol. 33: 561-563. Linsley,E. G. andJ. A. chemsak.1984. The cerambycidaeof North America,part vII, no. l: Taxonomyand classificationof the subfamily Lamiinae,tribes Parmeninithrough Acauthoderini.Univ. Calif. Publ.Entomol. Noguera,F. A. 1993. Revisi6n taxon6micadel gdnero oncideres Serville en Mdxico (Coleoptera:Cerambycidae). Folia Entomol.Mex. 88: 9-60. Noguera,F. A., S. Zuagoza-Caballero,J. A. Chemsak,A. Rodriguez-palafox,E. Ramirez, E. Gonzalez-Soriano,and R. Ayala, R. 2002 Diversityof the family Cerambycidae (coleoptera) of the tropical dry forest of Mexico. I. sierra de Huautla, Morelos. Ann.Entomol. Soc. Am. 95:617-627. Rice, M. E. 1986.Winter mortality of Onciderespustulatus (Coleoptera: Cerambycidae) larvaeinduced by freezingtemperatures. J. Kans.Entomol. Soc. 59: 423-427. Rice, M. E. 1989. Branch girdling and oviposition biology of oncideres pustulatus (Coleoptera:Cerambycidae) on Acaciafarnesiana. Ann. Entomol. Soc. Am. 82: 181-186. 3il AUTHORINDEX TO VOLUME 29 Ar6valo-Niio, Katiushka,I 53 McAllister,C.D.,263 Austin,James W., I Mejia-Ford,O.L, 185 Bextine,Blake R.,47 Mitchell, ForestL., 13 Birdsall,Stephen L., 23 Mohammed,A. Al-Doghaid,6l Bischoff,K.P.,263 Morales-Ramos,Lilia H., 153 Boroda,Eli,55 Muegge,Mar:kA.,277 Borth,Leahann M.,ll7 Natw'ick,EricT,23 Bossard,R.L.,271 Olson,J.K., 185 Brain,Matthew S.,77 Payton,Mark, 99 Bundy,C.S.,305 Phillips,P.A., 175 Chen,T.-Y., lll Rao,MaheshN.,227 Chu,C.C., lll Reagan,T.E.,263 Chu,Chang-Chi,83 Renouard,J.J., 9l Claassen,M,,209 Richman,D.B.,9l Coleman,R.J.,69 Roberts,B. Wanen,99 Cook,JerryL.,77A Rodriguez-del-Bosque,L.A., 69. 309 Cook,Tamara J., 77 Roozeboom,K.,209 Cooper,Dayna D., 39 Rosas-Garcia,Ninfa M., 153 Cortez-Mondaca,E.,69 Royal,T.A,, 31,245 Cranshaw,Whitney, 39, 6l Sanson,D.W.,271 Creamer,R., 9l Schexnayder,H.P., Jr.,263 Dogramaci,Mahmut,99 Schmidt,Justin O.,253 Dreslippe,Richard J., 117 S6tamou,Mamoudou, 137 Edelson,J.V.,99 Shapiro,JeffreyP., 301 Elliott, NormanC., 227, 245 Showler,Allan T., 137 Elzen,G.W.,l47 Shrefler,Jim W., 99 Evans,P.,209 Shufran,R., 167 Fleenor,Scott B., 297 Singh,J., 167 Foil,L.D.,27l Smith,DudleyT.,285 Foster,Bart T., I Sokolova,Y.Y.,271 Fuxa,J.R.,271 Staten,Robert T.,23 Galan-Wong,Luis J., 153 Szalanski,Allen L., I Giles,Kris, 245 Taber,Stephen W .,297 Gold, RogerE., I Taylor,Merritt J., 99 Gravois,K.A.,263 Thorvilson,Harlan G., 47, 55 Guerero,Felix D., 193 Vargas-Camplis,J.,69 Hanna,Anita,253 Walker,N.R., 3l Harvey,T.,209 Way,M.O., 185 Henneberry,T.J.,lll Wenhua,Lu,99 Hesler,Louis, 245 Whitworth,J., 167 Janssen,K,,209 Wilde,G., 167,209 Kakischuk-Tymensen,Lisa, 193 witt, M.,209 Kindler, S. Dean,227, 245 Yang,Zhiming,227 Knutson,Allen 8., l3 Yee,W.L., 175 kgaspi, JesusaCrisostomo, 301 Legaspi,Jr., Benjamin C., 301 Leyva-V6zquez,J.L., 69 Liu, Tong-Xian,83,727 Long,J,,209 Lysyk,Tim J., 193 313 SUBJECTINDEX TO VOLUME29 Agrotis ipsilon, Diabrotica virgifera vilgifera, survivalof larvaeof and useofthe Perocon@CRM trapto observationson larvalpredation monitorabundance of. 167 by thief ant in turfgrass,3 I Diatraeasaccharalis, Aphis gossypii, evaluationof feedingstimulants hydrocarbonsof greenand yellow combinedwith polymersto colorsmorphs of, I 17 developformulations against, 153 Apis mellifera, transgenicB l-com affectson, 263 effectof phagostimulantsin artificial dietsfor,253 Exoprosopafasciata, in centralTexas,297 Babesiatabaci- responseofto cool white fluorescent Haematobiairriatans, light,lll expressedsequence tags and new laboratorytoxicify of insecticide genecoding regions from, 193 residuesto, 147 Hibana incursa, Beauveriabassiana, gut contentanalysis ofusing deliverysystem for biologicalcontrol serologicalmethods, 9 I of thered importedfrr.e ant,47 Mecidea, B lis susI eucop terus Ieucop I erus, in New Mexico,305 comparativebiology of in rice and sorghum,185 Onciderespustulata, an outbreakofin northem Cabbage, Tamaulipas,Mexico, 209 effectof interplantingof necteriferousplants on pestsof, Peanuts, 6l pest managementpractices for in the southeastemUnited States, 285 Ceratitis capitata, differential mortality of natural Pe c tin op ho ra gossypi e I Ia, enemiesof exposedto malathion- evaluationof cottonstalk destruction treatedleaves during an for controlof. 23 eradicationpro gram, l7 5 Po disus maculiventris, Catolaccusgrandis, biochemicalcomparisons of field effectsofkaolin particlefilm on andlaboratory populations of, selectedarthropod populations in 301 cotton,137 evaluationof for boll weevil control Reticulitermes, in Mexico,69 geneticvariation and geographical distributionof in Texas,I Cotton, evaluationof stalk destructionfor Rhopalo sip hum rufi ab d o minal is, controlof pink bollworm,23 seasonalabundance ofin wheat,245 314 Schizaphisgraminum, remotesensing to detectplant stress cawedby,227 Solenopsisinvicta, damageto peanut,13 infectionof by Thelohania solenopsae,TT mortalityof by castorseeds, 55 themicrospori di;m, TheIo hani a solenopsae,in, 27| Solenopsismolesta, predationby on larval black cutworm,3l Sorghum, Seedtreatment for controlof insect pestsof,209 Thripstabaci, comparisonof absoluteestimates of with visualcounting and sticky traps,83 seasonalpopulation dynamics, life stagecomposition, and predaceousnatural enemies ofon onions,llT Toumeyella, seasonalbiology and natural enemies of, 39 Trialeuro des abuti Ione am, responseofto cool white fluorescent light,lll TriaI eurodesvaporari orum, responseofto cool white fluorescent light,lll Trichoplusiani, Developmentof a sequential samplingplan for,277 Watermelon, occurrenceand abundance of arthropodpest of in Oklahoma, 99 315 t_ Unled Statg! Postal SlMce Statementof Ownershlp,Management, and Glrculailon Southreatera Bnt@ologLst Xrrch, J|De, SepteDber, Ileccuber !99f"ty of Southreateru EatoDloglsts, c/o Allea llrtro U360 Coit noad, Ilallaa, fX ISZSZ of lg:1-9q SouthnBsteE! BDtmloe'.sts, c/o lllet XDaraor 17350 Colt load, Ilallas, tX. ISZS| 9gglty of Southreater Entoulogtsts, c/o Allea fauraon 17360 Gott foad, Irrlla6, tX tSZSi Darrell gay, addreea above t0. Owrs @ ,tMbythe Mrct ed,tdd'aca ffi,glbilE @ch lndlvuul one. @rcllttth@ol It|uF. dr€d(bd Td SbtF {Fd by aqwolL TIE Duesa. lr ana'ne ermpir;tsG ffri":T"i"il"*X$"XfJilH,f i? ffiffii"aniatron E Has Chrnged Owfng PE€dtng 12 t onths (A/bflrrrer h6t Nbril exp!&ilon d dwtrle $nh l'ils rt tffit) PS Fom 3526, Octobs t99o ,ngtucl;ffiqRol6e) 319 Tltle bsue Drlg ft. Cinlltim Oat. Below Av€rrg. ]{o. Coplt. Elch l.tu.' ilo. Copl.. .t slnll,e |3$b Ertill .nd t$tue of ClGuh0on burlno PEedino 12 lloitht Publhhed Nclnrt io Flllng D.te a. Totaf Numb€r of Cop|6 (N.t p@s M) yn 500 Pr|dnrqu$tod Ouhide.CounlyMall Subtcdpli@s sLlsd on 1 Fffi?1A|. (lrdttdc .dvaditelt Foot frd er.l'agp aelet) 286 P.H lFodnty Subsdlptlons Stal€d on Fom 35{l b. Pall and/or (hdtlde ailedlse/s [email protected] edvnge @ilest o Req@led CkEulatlon Salo.lrqgh D€sleF {d C€tiil, Stwt Wndgc, Colr{s Sd$, and Olh€t NGUSPS Psid Dsltlbr|fd 0 0 othar Cl.$€ M.llod Through lhe usPs 80 a7 c. Total P.id antt/o. R4slied Ciwlgtid lsun ot 15D. (1), (2),(3),a!i (4)l 365 351 FEe t1 Outrltecdnty s Stabd s Fm 3541 Okkiblilon o by M.ll (Sehpres, 12 lcCdnt.s Sbl€d m Fom 354'l 0 o ao4 ffi dlwft8) I3 olhr Cb$e. ilailed Th60gh th€ USPS o 0 e. F@ Di6ldbullon Ortdd€ lhe Mr, (Cailb6 6 olher tuN) 5 8 Toi6f F@ Ofstihi{on /Sufr d l&. ed 15e.) 6 8 9 To,"f Di",rlbu,i* 1s- ol 15c..rt tst) 372 359 CoDles nol Dlslribdod t28 141 '' Iolal (Sun of tag- frd h't 500 j. Percent Psld and/q R.q$sled CLculation 97.8 (15c" dwd by 159. dM 1@t 98.4 El Publicallon bs oltil3 O Plbileti0 not f en fy rm * fr'omt-o ftf,niBh€d on lhis tom is lrue and Mplele. I undeEbnd that uyse who tumlahs t'bs d whd dtt! mde,ial d htrormton EqFsted o fE rm my b6 sbt€ai to crhnd $ndids (indudlnC fims td andd civil 3adion3 lnstructions to Publlshers complgte and file one 6py of thls 'om wlth your postmster amually on or beio€ octobq 1. Ksp I @py oFlh€ @mdeled bm for !@urre@rds. 'l In whsc lhe stckholder ot seddty hoHel is a truslse, includ€in it'ams 0 and 1I the nare ol lhs peBon tr.@rpoEliof, ior €ses pft€nt wt om itte trusfe is aaing. a|$ imtuOetie mffi and addc$es of indMduls who aG stockholdeFwho m or hold 1 or mF of th€ total amu-nt ot bonds,mort969es, or olher seddties of the publishingoPoration. In item 1I , if mne, chek the box.Use blanksheeis lf rcE spae is requlred. Be sue to fumish all circulalim inio|mtion caltsdior in item 15. Freecirculat'pn must be shom io ltems 15d,e. md f. Etum€dto the Item 15h.,Copi6 'eturns not Dlstrlbuted,must include (1) newstand@pis diginallyBtated on Fom 3541.8nd Publisher' (Zj esnmjteO trcm nem agents,and (3); dopiesfor ofri@ Be, leftoverc.spoiled, and all other@pies rct dlstributed. lf th€ publiBtion had P€rlodicalsauthoriailon as 6 €€reEt or requ6_t6rpubli€tbn, this statemEnlof oreFhlP, lranag€ment, i,iij-Ciruijti"n r"ii Ue puUti"tr"O;it .uit Ue priniei In my issue ln Octoberor, if the publicstionls rct publishsdduting October' lhe first lssu€ printedatler Ociobet o. ln item 16. indi€ts the date of the issue In whidr this Statemnt of OwreEhip will be publlshed' Item l7 mustbe slgned. Fattun to lito q pubnth t shtement ol omeBhtp may tead to suapereion ol Periodlcal' auahottzilbn. PS Fom 3526, october 1999 (R€86e, 320